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385 publications mentioning hsa-mir-31 (showing top 100)

Open access articles that are associated with the species Homo sapiens and mention the gene name mir-31. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

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[+] score: 493
DEN -induced rat HCC tissues showed the upregulated expression levels of HDAC2, CDK2, cyclin D1, cyclin A, N-cadherin, fibronectin and suppressed expression levels of p21 [WAF1/Cip1] and E-cadherin accompanied by reduced-miR31 expression compared to normal rat hepatic tissues (Fig. 5B and C). [score:11]
Our results showed inhibition of DNA methylation by either 5-aza-dC treatment or knockdown of DNMT1 caused the induction of miR-31 expression, and consequently suppressed HDAC2 and CDK2 expression in liver cancer cells. [score:10]
Therefore, we hypothesized that some cancer-driver genes targeted by miR-31 are up-regulated in HCC as miR-31 was down-regulated in HCC. [score:9]
MiR-31 was significantly down-regulated in overt HCC and ectopic expression of miR-31 mimics inhibited in vitro tumor growth and metastatic ability in liver cancer cell lines. [score:8]
In this study, we showed that miR-31 expression was significantly down-regulated in a subset of HCCs, and the low expression of miR-31 was associated with a poor prognosis in HCC patients. [score:8]
As a tumor suppressive role of miR-31 in tumorigenesis, it was reported that miR-31 directly or indirectly controls expressions of specific proteins involving hallmarks of cancers, such as cell cycle, apoptosis, migration and cytoskeleton regulating molecules. [score:8]
The fact that HDAC2 and CDK2 are up-regulated in HCC led us to hypothesize that normal HDAC2 and CDK2 expressions are balanced by endogenous miR-31, which selectively controls HDAC2 and CDK2 mRNA translation in normal hepatic liver cells. [score:8]
Disruption of DNA methylation by either 5-aza-dC treatment or DNMT1 knockdown caused the induction of miR-31 expression, and thereby suppressed HDAC2 and CDK2 expression in both SNU-449 and SKHep-1cells (Fig. 6D and E). [score:8]
In our study, western blot analysis showed that HDAC2 and CDK2 protein levels were decreased after ectopic expression of miR-31, and simultaneously induced p21 [WAF1/Cip1] and suppressed the expression of cyclin A and cyclin D in both SNU-449 and SKHep-1 cells. [score:7]
For liver cancer, one recent study reported that miR-21, miR-31, miR-122, miR-221, miR-222 were significantly up-regulated in HCC tissues, whereas miR-145, miR-146a, miR-200c, and miR-223 were found to be down-regulated [15]. [score:7]
Furthermore, in prostate cancer, miR-31 was identified to negatively regulate E2F1, E2F2, EXO1, FOXM1, and MCM2, which are the key regulatory proteins in cell cycle regulation, and thereby demonstrated that the downregulation of miR-31 disrupts cellular homeostasis and contributes to the evolution and progression of prostate cancer. [score:7]
Therefore, to validate the expression of miR-31 in liver cancer, we observed miR-31 expression in the large cohorts of HCC patients available from the National Center for Biotechnology Information (NCBI) and Gene Expression Omnibus (GEO) database (accession numbers GSE21362 and GSE39678), and the data were presented as scatter plots. [score:7]
Taken together, we present evidences that miR-31 functions as a tumor suppressive miRNA by directly regulating HDAC2 and CDK2 expression in liver cancer progression. [score:7]
For example, miR-31 was demonstrated that overexpression of miR-31 led to increased growth rate by targeting suppressors, LATS2 and PPP2R2A in lung cancer [22]. [score:7]
Although we showed that ectopic miR-31 elicited inhibition of cellular growth of liver cancer cells through targeting HDAC2, it is necessary to prove that ectopic overexpression of 3′ UTR- deleted HDAC2 plasmid (pME18s-HDAC2-FLAG) can rescue the effects on cell cycle molecules in the same cells. [score:7]
Our results showed that ectopic expression of miR-31 resulted in suppression of HDAC2 and CDK2 protein expression in liver cancer cells. [score:7]
These results suggest that the expression of miR-31 is suppressed in HCC and its low expression associates with biological process of tumorigenesis and poor prognostic signs of HCC patients. [score:7]
Expression of miR-31 correlates inversely with breast cancer progression in humans, where an increase in expression of miR-31 target genes was observed as tumors progressed to more aggressive forms. [score:7]
Although the underlying mechanism leading to the suppression of miR-31 should be clearly elucidated, these epigenetic alterations cause the down-regulation of miR-31 and thereby contribute to the hepatocellular malignant transformation and proliferation. [score:6]
For instance, miR-31 was significantly down-regulated in breast cancer and bladder cancer, thus expression of miR-31 was inversely correlated with metastasis and aggressiveness [10, 11]. [score:6]
To clarify that expression of miR-31 is regulated by EZH2, cells were treated with DZNep (3-Deazaneplanocin A, an inhibitor of S-adenosylmethionine -dependent methyltransferase, and stimulates degradation of EZH2). [score:6]
It was found that miR-31 was able to suppress reporter gene activity in these cells, whereas mutants plasmids showed no changes in the reporter gene activity in both SNU-449 and SKHep-1 cells indicating miR-31 selectively regulate both HDAC2 and CDK2 expressions in HCC cells in vitro (Fig. 2D). [score:6]
Notably, we also observed that targeted-disruption of HDAC2 recapitulated the effect of ectopic miR-31 expression on the same molecules whereas CDK2 knockdown did not affect. [score:6]
Earlier report showed that miR-31 is located at the chromosome 9q21.3, and this locus is very close to the locations of tumor suppressors, CDKN2A and CDKN2B, that is frequently deleted in many cases of cancers, which may result in down-regulation of miR-31 [27, 28]. [score:6]
Unlikely with previous observation in liver cancer study, miR-31 expression was significantly down-regulated in these two different HCC cohorts (Fig. 1A). [score:6]
Our data, contradictive with previous observation, indicated that miR-31 expression was significantly down-regulated in patients with HCC. [score:6]
Next, to better understand the underlying mechanism of the growth inhibition elicited by miR-31, western blot analysis was performed for cell cycle regulatory proteins and miR-31 -targeting molecules, HDAC2 and CDK2. [score:6]
Ectopic expression of miR-31 elicits a tumor-suppressor effect by regulating cell-cycle proteins in liver cancer cells. [score:6]
MiR-31 inhibits liver cancer cell growth by targeting G1/S transition regulatory molecules. [score:5]
Figure 3(A) Ectopic expression of miR-31 suppressed SNU-449 and SKHep-1 cell proliferation. [score:5]
The direct target molecules of miR-31, HDAC2 and CDK2, as well as the cell cycle regulators and EMT markers, were analyzed with immunoblotting. [score:5]
In addition, Kaplan-Meier survival curves of patients with HCC indicated that the 5-year overall survival (OS) rates of HCC patients with low miR-31 expression was significantly lower than that of HCC patients with high miR-31 expression (Fig. 1B). [score:5]
As shown in Fig. 2B, Dicer knockdown augmented HDAC2 and CDK2 protein expressions in SNU-449 and SKHep-1 cells, whereas co-transfection of miR-31 mimics attenuated Dicer knockdown effect on the same cells. [score:5]
Interestingly when we performed prediction analysis of putative miR-31 targets by using miRWALK database, 399 genes were resulted in possible targets of miR-31. [score:5]
MiR-31 is well known metastatic suppressor by direct targeting integrin family, RhoA and RDX in various cancers but unknown in liver cancer [14]. [score:5]
Since EZH2, a core component of polycomb repressive complex2 (PRC2), was reported to be over-expressed in HCC, we assumed that hyper-methylation of H3 Lys-27 residue may be related with the suppression of miR-31 [20]. [score:5]
Notably, treatment of DZNep elicited remarkable suppression of EZH2, HDAC2 and CDK2 proteins with concomitant increase of miR-31 expression in SNU-449 and SKHep-1 cells (Fig. 6B and C). [score:5]
The phenotype caused by aberrant miR-31 expression seems to be strongly dependent on the endogenous expression levels. [score:5]
MiR-31 is aberrantly down-regulated in HCC and its expression is associated with the poor prognosis of patients with HCC. [score:5]
In colorectal cancer, it was demonstrated that miR-31 plays a significant role in activating the RAS signaling pathway through the inhibition of RASA1 translation, thereby improving colorectal cancer cell growth and stimulating tumorigenesis [24]. [score:5]
Thus, suppressive function of miR-31 on EMT molecules seems to be indirect effect of miR-31, and HDAC2 may also contribute to selective regulation of these EMT molecules. [score:5]
In the present study, we demonstrated that miR-31 functions as a tumor suppressor in the development of HCC by negative regulation of the major components in the cell cycle transition and EMT processing of cancer cells. [score:5]
Notably we also found that treatment of DZNep elicited remarkable suppression of EZH2, HDAC2 and CDK2 proteins with concomitant increase of miR-31 expression in liver cancer cells (Fig. 6B-E). [score:5]
Thus, to identify miR-31 target genes, we used the target prediction program, miRWALK (http://www. [score:5]
Notably we also able to generalize the repressive status of miR-31 expression in HCC by recapitulating miR-31 expression from the various large cohorts of HCC patients that are available from the NCBI, GEO database (Fig. 1). [score:5]
Ectopic expression of miR-31 potentially suppressed cell growth via transcriptional inactivation of HDAC2 and CDK2. [score:5]
The liver cancer cell lines, SNU-449 and SKHep-1, were treated with indicated drug (01% DMSO or 10 μM of 5-aza-dC), or transfected with siRNA (negative control siRNA, si- DNMT1), and analyzed protein expressions of miR-31 target genes, HDAC2 and CDK2. [score:5]
These results demonstrate that miR-31 is a direct regulator of endogenous expression HDAC2 and CDK2 in liver cancer cells. [score:5]
Ectopic expression of miR-31 increased the oncogenic potential of head and neck squamous cell carcinoma cells under normoxic conditions in cell culture or tumor xenografts by impeding factor-inhibiting hypoxia-inducible factor [23]. [score:5]
For example, miR-31 down-regulation has been detected in several other malignancies, such as bladder, esophageal, ovarian, and prostate cancer as well as in glioma, leukemia, melanoma, and mesothelioma. [score:4]
Here we report that miR-31 functions as a tumor suppressor through the regulation of cell cycle and epithelial-mesenchymal transition (EMT) proteins in hepatocarcinogenesis. [score:4]
MiR-31 regulates metastatic potential of liver cancer cells and is suppressed by deregulation of epigenetic modifiers. [score:4]
From this, all 9 HCC tissues exhibited significantly down-regulation of miR-31 in HCC (Fig. 1C). [score:4]
In addition to breast cancer, not only that miR-31 acted as a metastatic suppressor by regulating these genes, but also elicited cell cycle arrest and apoptosis in lung cancer [25]. [score:4]
si- HDAC2 and si- CDK2 were used for knockdown of miR-31 target genes, respectively. [score:4]
Thus, to support our hypothesis that HDAC2 and CDK2 expressions are regulated by miR-31 in HCC cell lines, we introduced Dicer specific siRNAs to block miRNA biogenesis in HCC cells. [score:4]
The anti-growth effect could be partially explained by the disruption of cell growth regulation on miR-31 targeting, such as cell cycle arrest, cellular senescence or apoptosis. [score:4]
These results demonstrate that miR-31 regulates cell cycle molecules through the selective control of HDAC2 expression in liver cancer cells. [score:4]
Next, to verify that miR-31 specifically binds to 3′UTRs of CDK2 and HDAC2 to interfere translation of those transcripts, mutant vectors harboring random mutation sequences of miR-31 biding sites of the 3′UTR of CDK2 and HDAC2 genes were generated, and then each vector was co -transfected with miR-31 into SNU-449 and SKHep-1 cells. [score:4]
A modified Boyden chamber assays revealed that ectopic expression of miR-31 mimics significantly suppressed chemoattractant (5% fetal bovine serum)-stimulated migratory and invasive responses of both SNU-449 and SKHep-1 cells, whereas AS-miR-31 co-transfection significantly rescued anti-migratory and invasion effects in the same cells (Fig. 4A and B). [score:4]
Thus, we next explored the effects of miR-31 overexpression on cell death and cell cycle regulation. [score:4]
Interestingly, in a subset of HCCs defined by Edmondson grade I (TG1, n = 5), grade II (TG2, n = 5), grade III (TG3, n = 6), miR-31 was gradually down-regulated in the progression of liver cancer (Fig. 1A, GSE39678). [score:4]
In another study, it was identified that WAVE3, actin cytoskeleton regulating molecule, was shown to be directly regulated by miR-31 [26]. [score:4]
Hence, the functional role of miR-31 is extremely complex and miR-31 can hold both tumor suppressive and oncogenic roles in different tumor types. [score:3]
MiR-31 is among the most frequently altered miRNAs in human cancers and altered expression of miR-31 has been detected in a large variety of tumor types. [score:3]
However, N-cadherin, E-cadherin, vimentin and fibronectin were not found as miR-31 target genes (data not shown). [score:3]
MiR-31 is down-regulated in hepatocellular carcinoma. [score:3]
In addition, overexpression of miR-31 mimics significantly abolished metastatic potential of HCC cells. [score:3]
Flow cytometric cell cycle analysis indicated that miR-31 overexpression led to an increase in the number of cells in the G1 phase with a concomitant decrease in the number of cells in the S phase and G2/M phase, but AS-miR-31 co-transfection attenuated this effect in the same cells (Fig. 3B). [score:3]
However, authors concluded that high level of miR-21, miR-31, miR-122, and miR-221 expression was correlated with cirrhosis but only miR-21 and miR-221 were associated with tumor stage. [score:3]
In some cases, such as colon cancer and oral cancer, miR-31 was highly over-expressed [12, 13]. [score:3]
In many different types of cancers, repressed miR-31 expression was demonstrated to contribute malignant transformation and proliferation of cancer cells. [score:3]
For liver cancer, the only one study reported that miR-31 was over-expressed, but no correlation with clinicopathlogical features was found [15]. [score:3]
In contrast, the suppressive effect of miR-31 on EMT molecules in liver cancer cells was significantly rescued by the con-transfection of AS-miR-31. [score:3]
The expression of miR-31 was normalized to U6 snRNA (* P<0.05; ** P<0.005, Student's t test) (D) The qRT-PCR analysis of miR-31 for hepatocellular carcinoma cell lines (n=7) and liver normal cell lines (n=2) (** P<0.005; *** P<0.001, Student's t test). [score:3]
To gain further insight into the regulatory effect of miR-31 on EMT, western blot analysis was performed for the EMT regulatory proteins in in liver cancer cells. [score:3]
In prostate cancer, hyper-methylation of miR-31 promoter was responsible for its low expression and contributed tumorigenesis [21]. [score:3]
MiR-31 regulates HDAC2 and CDK2 expression by binding 3′-UTR in hepatocellular carcinoma. [score:3]
Then, to determine whether HDAC2 and CDK2 are selectively regulated by miR-31 via direct interaction with the 3′-UTR of these genes, we cloned the 3′-UTR of HDAC2 and CDK2 into a reporter vector linking the luciferase open reading frame downstream to generate psi-CHECK2-HDAC2_3′-UTR and psiCHECK-CDK2_3′-UTR plasmid, respectively (Fig. 2C and Supplementary Fig. S1). [score:3]
Furthermore, even though expression of miR-31 and its functions were extensively studied and well defined in many cancers, the role of miR-31 in human liver cancer is still unidentified. [score:3]
The expression of miR-31 varies from one cancer to another and thus its functional role is very diverse in different malignancies. [score:3]
In contrast, overexpression of miR-31 in MIHA and L-O2 (immortalized normal hepatic cell lines) did not effect on cell growth rates of these two different cell lines (Supplementary Fig. S2). [score:3]
Inactivation mechanism of tumor suppressor miR-31 in liver cancer. [score:3]
The five year survival rate was significantly decreased in patient with low level of miR-31 expression in the tumor tissues (Log-rank P = 0.0015*) (C) The qRT-PCR analysis for 9 paired HCC tissues. [score:3]
These results provide the underlying mechanisms leading to the suppression of endogenous miR-31 in HCC. [score:3]
Thus, the aberrant expression of these miRNAs, such as miR-31 and -122 remained to be validated in liver cancer. [score:3]
On the contrary to this, miR-31 was also reported as a tumor suppressor in other type of cancers. [score:3]
In breast cancer, miR-31 was under expressed in metastatic tumor and negatively correlated with high risk of metastasis. [score:3]
However, increased expression of miR-31 has also been detected for example in colorectal, lung and pancreatic cancer, head and neck squamous cell carcinoma, and osteosarcoma [14]. [score:3]
From this database, at least in six out of eight different prediction programs, 399 genes were predicted to be targeted by miR-31 (data not shown). [score:3]
Next, to verify the suppression of miR-31 in HCC patients, miR-31 expressions of 9 randomly selected HCC tissues paired with adjacent non-cancerous liver tissues were investigated by quantitative real-time PCR (qRT-PCR). [score:3]
Our results demonstrated that repression of miR-31 contributes to transcriptional activation of HDAC2, and, thereby causes the acceleration of cell cycle transition of cancer cells through selective regulation of cell cycle components (Fig. 3). [score:2]
Next, to investigate biological functions of miR-31 in hepatocellular malignant proliferation and transformation, we attempted ectopic expression of miR-31 and studied in the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenytetrazolium bromide (MTT) assay for the measurement of cell growth rate of two different liver cancer cell lines, SNU-449 and SKHep-1. Ectopic overexpression of miR-31 resulted in reduced growth rates of these two different liver cancer cell lines, whereas co-transfection with AS-miR-31 (an antisense inhibitor of miR-31) significantly blocked this anti-growth effect (Fig. 3A). [score:2]
In vivo validation of miR-31 regulating molecules in DEN -induced rat liver cancer mo del. [score:2]
Figure 5 In vivo validation of miR-31 regulating molecules in DEN -induced rat liver cancer mo del(A) Macroscopic observation of the whole liver from the DEN -induced rat mo del (arrows; tumors). [score:2]
MiR-31 is one of well identified miRNAs in cancer biology, and interestingly, regulation patterns and functions of miR-31 were diverse depending on cancer types. [score:2]
However, miR-31 overexpression showed no significant induction of apoptotic cells compared to miRNA control (Fig. 3C). [score:2]
Although further research is required to identify regulatory mechanisms for the repression of miR-31 in liver cancer, the results suggest that the miR-31 may play a central role in hepatocellular malignant transformation and proliferation providing novel therapeutic intervention of liver malignancy. [score:2]
MiR-31 was significantly down-regulated compared to corresponding non-tumor tissue. [score:2]
For mutagenesis of the miR-31 -binding site, a QuickChange site-directed Mutagenesis Kit (Agilent Technologies, Palo Alto, CA) was used according to the manufacturer's instructions. [score:2]
These results suggest that anti-metastatic potential of miR-31 could be attributed to the selective regulation of EMT proteins in liver cancer cells. [score:2]
The human liver cancer cell lines (Hep3B, Huh7, PLC/PRF/5, SK-Hep-1, SNU-182 and SNU-449) exhibited relatively low miR-31 expression levels compared to that of non-cancer cell lines (MIHA and L-O2). [score:2]
In parallel with our previous observation, miR-31 selectively regulated EMT proteins, N-cadherin, E-cadherin, vimentin and fibronectin, to control metastatic potential of liver cancer cells (Fig. 4). [score:2]
MiR-31 suppressed motility and invasion of HCC cells. [score:2]
From this, obviously miR-31 is appeared to be suppressed in HCC compared with that of non-cancerous surrounding tissues. [score:2]
Wild type or mutant 3′-UTR construct of HDAC2 and CDK2 were cloned into a psi-CHECK2 vector, respectively, and co -transfected with miR-31 mimics in SNU-449 and SKHep-1 cells. [score:1]
The levels of HDAC2 and CDK2 in the Bio-miR-31 pull-down were quantified by qRT-PCR. [score:1]
Transfection of antisense miR-31 (AS-miR-31) attenuated anti-growth effect of miR-31. [score:1]
We then stained the cells with annexin V-FITC and PI after transfection of miR-31 mimics for apoptosis analysis. [score:1]
In addition, to clarify the direct interaction between miR-31 and 3′-UTRs of the two transcripts, we carried out biotin-labeled RNA pull-down assays. [score:1]
In similar, when ras-transformed NIH-3T3 cells were transfected with miR-31 mimics to generalize the effect of miR-31 in the regulation of metastatic potential, we obtained consistent results in both motility and invasion assays (Supplementary Fig. S3). [score:1]
Notably, N-cadherin, vimentin and fibronectin, hallmarks of EMT, were dramatically decreased in miR-31 mimics transfectants, whereas E-cadherin, an epithelial markers, was increased in both SNU-449 and SKHep-1 cells (Fig. 4C and D). [score:1]
Figure 4(A) of liver cancer cells transfected with miR-31 or co -transfected miR-31 with AS-miR-31. [score:1]
Thus, it is assumed that miR-31 has a specific function in each type of malignancy, and several mechanisms, including methylation -dependent silencing and local deletion, may explain its different roles in different tumor types. [score:1]
Small interfering RNAs (siRNAs) of Dicer (sense: 5′-UAAAGUAGCUGGAAUGAUG-3′, antisense: 5′-CAUCAUUCCAGCUACUUUA-3′), CDK2 (sense: 5′-GGAGCUUGUUAUCGCAAAU, antisense: 5′-AUUUGCGAUAACAAGCUCC-3′), DNMT1 (sense: 5′-CACUGGUUCUGCGCUGGGA-3′, antisense: 5′-UCCCAGCGAGAACCAGUG-3′) and microRNA mimics of miR-31 (sense: 5′-AGGCAAGAUCUGGCAUAGCU-3′, antisense: 5′-AGCUAUGCCAGAUCUUGCCU-3′) were synthesized by Genolution (Seoul, Korea). [score:1]
In contrast, this result was significantly attenuated by the co-transfection of AS-miR-31 in the same cells (Fig. 3D). [score:1]
However, little is known about the miR-31 status in patients with HCC and the possible roles in hepatocarcinogenesis. [score:1]
SNU-449 and SKHep-1 cells were transfected with miR-31 mimics after transfected with Dicer siRNA or negative control siRNA (N. C). [score:1]
In previous studies, miR-31 was reported as an oncomir in several human cancers. [score:1]
On the other hand, hyper-methylation of miR-31 promoter region was also reported as inactivating mechanism for miR-31 in prostate cancer [21]. [score:1]
Thus, functional role of miR-31 in liver cancer is elusive and to be uncovered. [score:1]
SNU-449 and SKHep-1 cells were transfected with Biotin-labeled microRNA control (Bio-N. C) or Biotin-labeled miR-31 mimics for 48 hours. [score:1]
Additionally, endogenous expression of miR-31 was investigated by qRT-PCR in nine different liver cell lines, including immortalized normal hepatic cell lines (Fig. 1D). [score:1]
To be specific, ectopic miR-31 repressed metastatic potential and this result was explained by miR-31 -mediated repression of ITGA5, RDX and RhoA [10]. [score:1]
SNU449 and SKHep-1 cells were transfected with Bio-miR-31 or Bio-miR-control in two 60mm dishes. [score:1]
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[+] score: 433
We, therefore, propose that one underlying mechanism by which miR-31 suppresses tumor cell invasion is by directly targeting SOX4 and indirectly targeting EZH2 and HDAC3. [score:9]
Esophageal cancer Oncogene MicroRNA miR-31 EZH2 SOX4 HDAC3 Epigenetics MicroRNAs (miRNAs) are a class of highly-conserved, noncoding 18-25- nucleotide RNAs that function as negative regulators of gene expression at the post-transcription level, binding to the 3′-untranslated region (3′-UTR) of mRNAs transcripts and targeting them for degradation [1]. [score:8]
Genetic and epigenetic loss of miR-31 is associated with EZH2 overexpression in melanoma [11], suggesting that miR-31 directly or indirectly regulates EZH2 expression. [score:8]
Inversely, in non-invasive tumor cells miR-31 targets SOX4, EZH2 and HDAC3 by direct and indirect means to inhibit tumor cell invasion (Figure  7D). [score:7]
Our results indicate that DNA methylation and Polycomb -mediated histone methylation both contribute to miR-31 silencing, since treatment with the EZH2 inhibitor, DZNep, and the DNMT inhibitor, AZA, enhanced miR-31 expression. [score:7]
We analyzed the reciprocal expression regulation of miR-31 and SOX4 in esophageal squamous and adenocarcinoma cell lines by qRT-PCR and Western blotting using overexpression and shRNA knock-down approaches. [score:7]
miR-31 directly targets SOX4 and indirectly targets EZH2 and HDAC3. [score:7]
As previously reported [11], our data confirm that miR-31 inhibits EZH2 expression (Figure  4C and D) whereas EZH1 expression was unchanged. [score:7]
Expression of miR-31 inhibits SOX4, EZH2 and HDAC3 expression. [score:7]
In line with these results, overexpression of miR-31 in FLO1 cells suppressed the expression of SOX4 at both the protein (Figure  4C) and mRNA level (Figure  4D). [score:7]
Taken together, these results demonstrate that SOX4 is a direct target of miR-31, while EZH2 and HDAC3 are indirect targets. [score:7]
We demonstrate that miR-31 is significantly decreased in invasive esophageal cancer cells, while upregulation of miR-31 inhibits growth, migration and invasion of esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) cell lines. [score:6]
Most importantly, miR-31 expression was significantly upregulated in both cell lines following SAHA treatment (Figure  3D). [score:6]
Depending on the cellular context, miR-31 may be up- or downregulated, acting as an oncogene or tumor suppressor, respectively. [score:6]
These reports emphasize the complexity of miR-31 -associated phenotypes and the need to better define miR-31 targets, as well as pathways regulating miR-31 expression in different cancers. [score:6]
Next, to focus on the biological significance and regulatory mechanisms of miR-31 expression in invasive adenocarcinoma and squamous cell carcinoma, we expressed miR-31 in invasive ESCC and EAC cell lines and analyzed the effects on cell migration and invasion. [score:6]
miR-31 expression is downregulated in invasive esophageal cancer cells. [score:6]
To analyze the regulation of miR-31, EZH2, EZH1 and HDAC3 by SOX4, we chose TE11 cells, which express low SOX4 and EZH2 levels, had high expression of miR-31 as a mo del. [score:6]
Conversely, miR-31 is upregulated in serum and tissue samples of esophageal squamous cell carcinoma (ESCC), with expression correlating to staging [18]. [score:6]
Overexpression of miR-31 has been linked to disease progression in colorectal cancer [5], head-and-neck squamous cell carcinoma (HNSCC) [6] and lung cancer [7]. [score:5]
DZNep treatment of TE8 and FLO1 cells resulted in a decrease in EZH2 expression and caused a dose -dependent increase in miR-31 expression in both of our invasive cell lines (Figure  3A, B, respectively). [score:5]
Yet, in another ESCC cohort miR-31 expression was decreased, and low miR-31 expression correlated with poorly differentiated tumors and decreased survival [19]. [score:5]
Ectopic expression of SOX4 in TE11 cells (Figure  4E) decreased miR-31 expression, although this was not statistically significant (Figure  4F). [score:5]
To determine the effect of PRC2 on miR-31 expression, we utilized the PRC2 inhibitor, 3-deazaneplanocin (DZNep). [score:5]
Loss of SOX4 led to a significant increase in miR-31 expression and strongly inhibited tumor cells proliferation, migration and invasion. [score:5]
Consistent with this observation, overexpression of EZH2 in TE11 cells (Figure  4G) led to a significant decrease in miR-31 expression (Figure  4H). [score:5]
Additionally, we show for the first time that histone deacetylation contributes to miR-31 silencing as treatment with the pan-HDAC inhibitor SAHA restored miR-31 expression. [score:5]
Hence, it is important to define not only the molecular pathways regulated by miR-31 but also the factors regulating miR-31 expression and functions in various tissues and cancers. [score:5]
SOX4, HDAC3 and EZH2 form a co-repressor complex to inhibit miR-31 expression. [score:5]
Pre-mature miR-31 expression was normalized to GAPDH and mature miR-31 expression was normalized to RNU6. [score:5]
Conversely, pharmacologic and genetic inhibition of SOX4 and EZH2 restore miR-31 expression. [score:5]
Figure 2 Ectopic expression of miR-31 suppresses migration and invasion of ESCC and EAC cell lines. [score:5]
TE8 and FLO1 cells, which express high levels of SOX4 and EZH2, showed the lowest expression level of miR-31. [score:5]
Furthermore, we observed a significant decrease in HDAC3 expression with miR-31 overexpression. [score:5]
We examined expression of EMT markers in miR-31 -overexpressing cell lines but found no significant alterations (data not shown). [score:5]
SOX4 knockdown (Figure  5A) using shRNA led to significant upregulation of miR-31 in TE8 and FLO1 cells, while miR-191 and miR-423-5p, used as controls, did not show any significant change (Figure  5B and C). [score:5]
Interestingly, EZH2 induced SOX4 expression by more than a 2-fold (Figure  4H), suggesting that not only does SOX4 regulate EZH2, but EZH2 also regulates SOX4 thereby potentially repressing miR-31 and/or other miRNAs and transcription factors. [score:5]
miR-31 has a known role in prostate cancer and melanoma, suppressing key cell cycle regulators and pro-oncogenic genes such as CDK1, E2F2, EXO1, FOXM1, MCM2, Src or MET [11]. [score:4]
Figure 1 miR-31 is downregulated in invasive esophageal cancer cells. [score:4]
miR-31 is downregulated in certain T-cell leukemias [8], breast cancer [9, 10], melanoma [11], ovarian cancer [12] and prostate cancer [13]. [score:4]
Based on data demonstrating epigenetic repression of miR-31 expression by DNA methylation and EZH2 -mediated H3K27me3 epigenetic mark in melanoma, leukemia and prostate cancer [8, 11], we examined the role of epigenetic regulation of miR-31 in invasive esophageal cancer. [score:4]
miR-31, in turn, targets SOX4 for degradation by directly binding to its 3′-UTR. [score:4]
Altogether, our results identify a feed-forward loop that leads to the activation of SOX4, which in turn up-regulates and binds to EZH2, cooperating with HDAC3 to repress the miR-31 promoter and advance esophageal tumorigenesis. [score:4]
Here, we show downregulation of miR-31 in invasive and aggressive esophageal cancer cells. [score:4]
Figure 7 SOX4, EZH2 and HDAC3 are upregulated and inversely correlate with miR-31 in esophageal cancers. [score:4]
Ectopic expression of miR-31 in ESCC and EAC cell lines leads to down regulation of SOX4, EZH2 and HDAC3. [score:4]
In FLO1 cells, however, miR-31 suppressed proliferation and colony formation (Figure  2D, E, respectively), indicating miR-31 regulates esophageal carcinoma cell growth in some cell lines. [score:4]
We identify SOX4 as a direct target of miR-31. [score:4]
A study by Asangani et al. recently showed that genetic and epigenetic loss of miR-31 leads to a feed forward upregulation of EZH2 [11]. [score:4]
Downregulation and loss of miR-31 in esophageal adenocarcinoma (EAC) correlates with poor patient prognosis [14- 16]. [score:4]
Our results indicate that miR-31 down-regulates SOX4 by binding to its 3′-UTR in EAC and ESCC cells. [score:4]
Additionally, miR-31 expression is reduced in EAC patients with poor histomorphologic response to neoadjuvant chemoradiation therapy [17]. [score:3]
Based on the observation that miR-31 targets both SOX4 and EZH2 and given that SOX4 binds to the EZH2 promoter to activate its transcription, we examined whether SOX4 leads to feed forward activation of EZH2, and subsequent miR-31 silencing. [score:3]
However, target prediction algorithms do not detect any putative binding site for miR-31 in the 5′UTR, 3′UTR or coding sequence of HDAC3. [score:3]
Additionally, miR-31 regulates EZH2 and HDAC3 indirectly. [score:3]
This may lead to tumor progression and the metastasis of EAC and ESCC; SOX4, EZH2, HDAC3 and miR-31 emerge as potential therapeutic targets. [score:3]
miR-31, miR-191, miR-423-5p expression was normalized to RNU6. [score:3]
These data suggest that miR-31 suppresses esophageal cancer cell motility and invasiveness, but cell growth depending on the cellular context. [score:3]
SOX4, EZH2 and HDAC3 levels inversely correlate with miR-31 expression in ESCC cell lines. [score:3]
Ectopic expression of miR-31 did not significantly affect tumor cell proliferation, causing only a marginal decrease in colony formation in the adenocarcinoma cell line FLO1. [score:3]
miR-31 expression was normalized to RNU6 and SOX4, EZH2, EZH1 and HDAC3 were normalized to GAPDH. [score:3]
miR-31 suppresses migration and invasion of aggressive ESCC and EAC cells. [score:3]
We conclude that miR-31 is not a strong inhibitor of EMT. [score:3]
After miR-200a and 200b, which are known for their roles in EMT, miR-31 was the most downregulated miRNA in invasive FLO1 cells compared to their less invasive OE33 counterparts by qPCR screen (Figure  1C). [score:3]
The lack of consistency in the literature with respect to miR-31 expression in esophageal cancer may be due to platform choice or normalization methods. [score:3]
Taken together, these data implicate SOX4 as a key mediator of the tumor-suppressive effects of miR-31 in this system. [score:3]
This suppressive effect was reversed by the four-nucleotide substitution in the miR-31 binding sequence. [score:3]
One day after plating, cells were transfected with the dual Renilla and Firefly luciferase reporter plasmid (psiCHECK-2) containing the full length 3′UTR of SOX4 (plasmid# 26989), the short WT oligo or mutant oligo along with a pBABE-miR-31 or pBABE Empty Vector expressing plasmid using FuGene HD (Promega). [score:3]
Ectopic expression of precursor and mature miR-31 in the respective cell lines was tested by quantitative RT-PCR (Figure  2A). [score:3]
Several mechanisms could contribute to aberrant miR-31 expression in cancer. [score:3]
We profiled SOX4, EZH2 and HDAC3 in ESCC (Figure  7A) and EAC (data not shown) cell lines and found a strong, inverse correlation between miR-31 and expression of SOX4, EZH2 and HDAC3 in invasive cancers of both histologies. [score:3]
Prior studies report that miR-31 expression is epigenetically silenced through promoter hypermethylation at CpG islands, as well as polycomb -mediated histone methylation [8, 11]. [score:3]
Similarly, miR-31 downregulation was observed in TE8 ESCC cell lines compared to TE11 (Figure  1D). [score:3]
We show that miR-31 significantly suppresses migration and invasion in vitro in both aggressive esophageal adenocarcinoma and squamous cell carcinoma. [score:3]
miR-31 expression is altered in multiple human cancers. [score:3]
Insert: correlation between mRNA expression of SOX4, EZH2, EZH1, HDAC3 and miR-31 in ESCC cell lines. [score:3]
Taken together, these data identify a molecular circuit where SOX4, EZH2 and HDAC3 target miR-31 to promote esophageal malignancy. [score:3]
Because SOX4 was recently shown to interact with other transcription factors, we tested whether SOX4 forms a co-repressor complex with EZH2 and HDAC3 to silence miR-31 expression. [score:3]
The WT OLIGO plasmid contained a 71-nucleotide region including the miR-31 target sequence. [score:3]
To test whether promoter methylation at CpG islands was involved in miR-31 silencing, we used the DNA methylation inhibitor 5′AZA-Deoxy-Cytidine (AZA). [score:3]
Using Western blot and qRT-PCR, we evaluated the effect of a pan-HDAC inhibitor (SAHA) on miR-31 expression. [score:3]
SOX4, EZH2 and HDAC3 inversely correlate with miR-31 expression in invasive esophageal cancer cells. [score:3]
Thus, we identified a novel molecular mechanism by which the SOX4, EZH2 and miR-31 circuit promotes tumor progression and potential therapeutic targets for invasive esophageal carcinomas. [score:3]
miR-31 expression was normalized to RNU6. [score:3]
We show that miR-31 is repressed in invasive esophageal cancers cell lines and that miR-31 levels inversely correlate with SOX4, EZH2 and HDAC3 expression. [score:3]
A sequence alignment search showed that the miR-31 target sequence in the SOX4 3′-UTR is conserved in humans and most great apes (Figure  4A). [score:3]
Treatment with AZA significantly increased the expression of miR-31 in TE8 cells and to a lesser extent in FLO1 cells (Figure  3E and F). [score:3]
Similarly, the suppressive effect of miR-31 on the SOX4 3′-UTR activity was observed in the esophageal tumor cell lines, TE8 and FLO1 (Figure  4B). [score:3]
To test whether SOX4 is regulated by miR-31 through direct binding to its 3′UTR, we used psiCHECK2 SOX4 full length 3′-UTR plasmid (WT) [3], and constructed two derivatives, SOX4 WT 3′-UTR oligo plasmid (WT OLIGO) and SOX4 mutant 3′-UTR oligo plasmid (MUT OLIGO) (Figure  4A). [score:3]
Likewise, miR-31 expression was higher in non-invasive cell lines such as the benign Barrett’s esophagus cell line CP-A compared to the metaplastic CP-B cell line (Figure  1E). [score:2]
SOX4 positively regulates EZH2, indicating a potential functional link between miR-31, EZH2 and SOX4. [score:2]
Furthermore, we confirmed the elevated miR-31 expression in OE33 cells, which have an epithelial phenotype, compared to FLO1 cells (Figure  1F). [score:2]
In line with this observation, Yamagishi et al. reported that PRC2 binds the miR-31 coding region and directly represses transcription of miR-31 in adult T-cell leukemia [8]. [score:2]
Consistent with previous reports [8, 11], we found that miR-31 negatively regulates EZH2 as well. [score:2]
miR-31 expression had no significant effect on proliferation in TE8 cells and did not alter the number of colonies in colony formation assays (Figure  2D, E). [score:2]
Here, we report that SOX4 promotes esophageal tumor cell proliferation and invasion by silencing miR-31 via activation and stabilization of a co-repressor complex with EZH2 and HDAC3. [score:1]
The pBABE-miR-31 plasmid (Plasmid#26088), pWPXL-SOX4 (plasmid#36984), pCMVHA-hEZH2 (plasmid#24230), Psicheck2 SOX4 full-length 3′UTR (Plasmid#26989) were purchased from Addgene (Cambridge, MA). [score:1]
Previous reports identify CpG islands and histone trimethylation mark sites upstream of the miR-31 promoter [8]. [score:1]
Based on the ChIP experiments, we propose that EZH2 and HDAC3 bind to a similar region on the miR-31 promoter, confirming that histone methylation and histone deacetylation contribute to miR-31 repression. [score:1]
Similarly, the mutant construct of SOX4 3′UTR (SOX4 Mutant OLIGO) which carried a substitution of four nucleotides within the core seed sequence of miR-31, was carried out using overlapping extension PCR and cloned between the XhoI and NotI site of the psicheck2 plasmid. [score:1]
The lower panel shows the nucleotide sequence alignment of the predicted miR-31 binding site in the 3′UTR of SOX4 of six species. [score:1]
These data suggest that PRC2, HDAC and DNA methylation are involved in miR-31 epigenetic silencing. [score:1]
To investigate the role of miR-31 in esophageal cancers, we examined the expression of miR-31 in ESCC, EAC and Barrett’s esophagus cell lines of differing invasive potential (Figure  1). [score:1]
TE8 and FLO1 cells were transfected with pre-miR-31 containing vector (grey bars) or empty vector control (black bars). [score:1]
We therefore speculated that loss of miR-31 in invasive esophageal cancer cells could be mediated, in part, by DNA and histone methylation. [score:1]
Finally, we tested whether SOX4, EZH2 and HDAC3 tethered to the miR-31 promoter. [score:1]
Studies on the role of miR-31 in esophageal tumors have produced conflicting results [16, 18, 19, 46]. [score:1]
We next investigated if loss of SOX4 functionally mimics overexpression of miR-31 in esophageal cancer cells. [score:1]
The upper panel shows the region containing the miR-31 binding site. [score:1]
ChIP analysis denoted H3K27me3 and HDAC3 enrichment in regions upstream of miR-31 (Figure  6C). [score:1]
Additional studies will aim to identify a subset of patients with concomitant high SOX4, EZH2 and HDAC3 and low miR-31 to demonstrate the mechanistic and clinical correlation between these pathways. [score:1]
With respect to miRNAs, prior work demonstrates that EZH2 interacts with AR to silence miR-31 in prostate cancers, and C-MYC recruits EZH2 to the miR-29 promoter in B-cell lymphomas [37]. [score:1]
HDAC3 and miR-31 in 16 ESCC cell lines. [score:1]
We show that EZH2 and HDAC3 bind to the miR-31 promoter using chromatin immunoprecipitation. [score:1]
org (maintained at cBio, the Computational Biology Center at Memorial Sloan-Kettering Cancer Center) predicted a miR-31 binding site (Figure  4A). [score:1]
However, we were unable to detect SOX4 at miR-31 promoter regions (data not shown). [score:1]
To examine the functional contribution of miR-31 in aggressive esophageal cancer, we transfected TE8 and FLO1 cells with vectors containing the precursor of miR-31 or an empty vector control. [score:1]
We conclude that SOX4, HDAC3 and EZH2 function as a potential co-repressor complex to silence miR-31. [score:1]
Despite the fact that our computational analysis did not detect any SOX4 regulatory elements upstream of the miR-31 promoter, co-IP assays show that SOX4 interacts with EZH2 and HDAC3. [score:1]
miR-31 is epigenetically repressed in invasive esophageal cancer cells. [score:1]
A SOX4 3′UTR fragment containing wild type (WT OLIGO) or mutant (MUT OLIGO) of the miR-31 -binding sequence was cloned into the downstream of the luciferase reporter gene. [score:1]
In this study, we explore the role of SOX4 and EZH2 in miR-31 repression and the contribution of miR-31 to survival, migration and invasion of aggressive esophageal cancers cells. [score:1]
We show that SOX4, EZH2 and HDAC3 form a co-repressor complex that binds to the miR-31 promoter, repressing miR-31 through an epigenetic mark by H3K27me3 and by histone acetylation. [score:1]
Taken together, our data identify a molecular circuit where SOX4, EZH2 and HDAC3 cooperate to repress miR-31. [score:1]
Interestingly, Valastyan et al. also reported that miR-31 promoted metastasis but not cell proliferation in breast cancer [9]. [score:1]
We hypothesized that SOX4 initiates the feed forward activation of EZH2, which in turn represses miR-31. [score:1]
Interestingly, miR-31 decreased HDAC3 on protein (Figure  4C) and mRNA levels (Figure  4D). [score:1]
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[+] score: 381
Other miRNAs from this paper: hsa-mir-21
As expected, the expression levels of Snail and two mesenchymal makers (N-cadherin and vimentin) were strikingly up-regulated in miR-31-overexpression cells, whereas SATB2 and two epithelial marker (E-cadherin and β-catenin) levels were down-regulated. [score:11]
This study clearly illustrated that miR-31 might directly regulate SATB2 expression by inducing mRNA degradation and translational suppression. [score:9]
miR-31-specific inhibitor transfection was employed to inhibit miR-31 expression in M5 and SW620 cells, which had high endogenous miR-31 expression. [score:9]
In summary, we observed that miR-31 expression was up-regulated in CRC, and such over -expression was significantly associated with aggressiveness of CRC patients, indicating poor clinical outcome. [score:8]
SATB2 was one of direct targets of miR-31 directly targeted SATB2 3’UTR. [score:7]
The down-regulation of miR-31 expression repressed aggressive phenotypes of CRC cells in vitro. [score:6]
0085353.g004 Figure 4The down-regulation of miR-31 expression repressed aggressive phenotypes of CRC cells in vitro. [score:6]
Clinicopathological analysis also found that high-level expression of miR-31 was significantly associated with lymph node metastasis and distant metastasis, which indicated that the up-regulation of miR-31 could be regarded as a predicted factor of metastasis for CRC patients. [score:6]
Several investigators reported that miR-31 up-regulated in CRC[10– 12] and squamous cell carcinoma of tongue[13], but down-regulated in breast cancer[14], gastric cancer[15], malignant mesothelioma[16] and pancreatic cancer[17] using qRT-PCR. [score:5]
Our results suggested that the up-regulation of miR-31 played an important role in CRC cell proliferation, invasion, and metastasis in vitro and in vivo through direct repressing SATB2, proving the miR-31 effect on tumorigenesis and progression. [score:5]
Thereafter, SATB2 over -expression could significantly attenuate the expression changes of the above markers associated with miR-31(Figure 7A). [score:5]
We observed that 75/143 (52.45%) CRCs had high-level expression miR-31, whereas 11/55 (20%) normal mucosa tissues had high-level expression of miR-31 (p<0.001). [score:5]
To search for potential targets of miR-31 that influence proliferation and migration ability of cells, we analyzed the putative miR-31 targets. [score:5]
CRC cells were infected with the recombinant lentivirus-transducing units plus 8 mg/ml Polybrene (Sigma, St Louis, Missouri, USA) and then subjected to FACS analysis for GFP expression to gain CRC cells with stable over -expression of miR-31. [score:5]
In addition, we found that the reintroduction of miR-31 into SW480 cells induced epithelial-mesenchymal transition (EMT), indicating the down -expression of epithelial markers and up -expression of the mesenchymal markers. [score:5]
We analyzed the changes of SATB2 expression in CRC cell lines after miR-31 over -expression. [score:5]
miR-31 and miR-21 can regulate their target genes as well as associated signaling pathways, and eventually enhance tumor aggressiveness. [score:4]
The luciferase activity of mutant reporters were unaffected by the simultaneously increased expression of miR-31 (Figure 5B), indicating that miR-31 directly bound to the 3’UTR of SATB2. [score:4]
miR-31 affected cell growth, migration, and invasion by directly targeting SATB2 in CRC. [score:4]
It was observed that the up-regulation of miR-31 in tumor samples was associated with lymph-node metastasis to a significant extent (p=0.0045, Figure 1B). [score:4]
Moreover, these tumors originating from SW480/miR-31 cells had decreased expression of SATB2, E-cadherin, and β-catenin, compared with that from SW480/miR-con (Figure 7B), suggesting that over -expression of miR-31 induced the EMT of SW480 cells both in vitro and in vivo. [score:4]
In this study, we explored the unambiguous role of miR-31 in CRC and found that the up-regulation of miR-31 was associated with the aggressive phenotypes of CRC and poor prognosis in patients. [score:4]
These findings suggested that SATB2 might be an important target of miR-31, wherein it regulated invasion and metastasis in CRC. [score:4]
miR-31 was up-regulated in metastatic CRC cells and human primary CRC tissues with lymph node metastases. [score:4]
SATB2 was a direct target of miR-31 in CRC cells. [score:4]
To further explore the mechanism by which miR-31 regulates tumor invasion and metastasis in CRC, we adopted some strategies that could identify miR-31 targets in CRC metastasis. [score:4]
However, it did not exhibit the direct evidence of EMT-related molecules that were regulated by miR-31/SATB2 in our studies. [score:3]
In this study, we also observed that the expression level of miR-31 in patients with lymph node metastases was higher than that in patients without metastases. [score:3]
The results revealed that high-level expression of miR-31 was correlated with short survival time of patients with CRC (51.85±3.78 vs. [score:3]
Over -expression of miR-31 induced CRC cells metastasis through EMT pathway. [score:3]
IHC staining showed that the tumors of control group displayed much lower Ki-67 index than that of miR-31 over -expression group (Figure 3B). [score:3]
The activities of SATB2 3’UTR luciferase reporter were responsive to miR-31 over -expression. [score:3]
Our experimental results further proved that SATB2 was a target of miR-31 in CRC cells. [score:3]
Previous studies have identified SATB2 as a target of miR-31 in cancer -associated fibroblasts[30]. [score:3]
The over -expression of SATB2 can significantly attenuate the promotion effects of miR-31 on cell proliferation. [score:3]
An increased expression of miR-31 significantly reduced the luciferase activity of the reporter containing SATB2 3’UTR in SW480 cells, but this did not affect the luciferase activity of the empty vector control (p<0.001, Figure 5B). [score:3]
They also identified that miR-31 suppressed breast cancer metastasis by a repressed cohort of pro-metastatic genes[14]. [score:3]
In patients with lymph node metastases, the relative mean expression of miR-31 was over 7.82 fold higher than that in patients without metastases (61.54±16.68 vs. [score:3]
These controversial results indicated that the role of miR-31 was possibly tumor specific and highly dependent on its targets in different cancer cells. [score:3]
The promotion of the proliferation and metastasis of CRC cells by miR-31 could be significantly attenuated by the ectopic over -expression of SATB2. [score:3]
For this purpose, SW480 cells with stable over -expression miR-31 (SW480/miR-31) and control cells (SW480/miR-con) were subcutaneously inoculated in nude mice. [score:3]
We observed a good negative correlation between miR-31 and SATB2 mRNA expression (Spearman's correlation analysis,  r = −0.687;  p=0.001, Figure 5E). [score:3]
The present study determines a positive role of miR-31 in carcinogenesis, invasion, and migration, via repressing SATB2 expression in CRC. [score:3]
In our study, we found that CRC patients with higher miR-31 expression tend to have advanced T-stage, lymph node or distant metastasis, which consistent with others’ findings in CRC. [score:3]
Furthermore, an over -expression of miR-31 was associated with shorter overall survival of CRC patients. [score:3]
Over -expression of miR-31 induced epithelial mesenchymal transfer in vitro and in vivo. [score:3]
Endogenous SATB2 expression in both mRNA and protein decreased in miR-31 precursor -transfected CRC cells. [score:3]
Two putative miR-31 binding sites at 3’UTR of SATB2 were site-directed and mutated, respectively using GeneTailor Site-Directed Mutagenesis System (Invitrogen). [score:3]
Indeed, SATB2 over -expression in SW480/miR-31 cells could inhibit both proliferation and migration ability by using CCK8, colony formation, wound-healing and matrigel invasion assays respectively, compared as that of SW480/miR-31 (p<0.05, Figure 6). [score:3]
In animal mo dels, the over -expression of miR-31 was sufficient to promote CRC cells metastasis to lung, liver, and organs in the peritoneal cavity. [score:3]
This was done to establish two stable miR-31-overexpression cell lines and two mock cell lines (miR-con). [score:3]
SW480/miR-31 cells with stable miR-31 over -expression were transfected with pCAG-SATB2 vector which encoded the entire SATB2 coding sequence but lacked the 3’UTR. [score:3]
Previous studies have revealed that the miR-31 expression was positively related to advanced TNM stage[10, 11] and/or deeper invasion of tumors[10] in CRC. [score:3]
To explore the potential biological function of miR-31 in CRC carcinogenesis and progression, we established two stable miR-31 over -expression cell lines to test the effect of miR-31 on behaviors of tumor cells. [score:3]
0085353.g007 Figure 7Over -expression of miR-31 induced epithelial mesenchymal transfer in vitro and in vivo. [score:3]
0085353.g001 Figure 1The levels of miR-31 expression in CRC cells and tissues by qRT-PCR or in situ hybridization and its prognostic value in patients with CRC. [score:3]
We first examined miR-31 expression by a stem-loop quantitative RT-PCR in a panel of CRC cell lines and 31 pairs of CRC and adjacent non-neoplastic mucosa tissues. [score:3]
org/Didier_Trono) to generate pLV-miR-31 expression vector. [score:3]
Inhibition of miR-31 reduced the growth, invasion, and migration of CRC cells in vitro. [score:3]
A multivariate Cox proportional hazard regression analysis revealed that miR-31 over -expression had a significantly worse prognostic impact on the overall survival of CRC patients independent of distant metastasis. [score:3]
miR-31 mimics and antisense inhibitors containing 2’-OMe (O-methyl) modifications were synthesized by GenePharma (Shanghai, China). [score:3]
As shown in Figure 2B-2D, the over -expression of miR-31 increased cancer cell proliferation and its ability to form colonies than that in the mock cells and wild type non-infected cells (p<0.001). [score:3]
An increased expression of miR-31 upon infection in two cell lines was confirmed by real-time RT-PCR (Figure 2A). [score:3]
The results showed that miR-31 was remarkably up-regulated in CRC tissues compared with adjacent non-neoplastic normal tissues (p=0.0004, Figure 1A). [score:3]
The expression level of miR-31 in the metastasis cells was higher than those cells with little metastatic powers. [score:3]
The animal experiment results showed that miR-31-overexpression SW480 cells exhibited dramatic metastasis of liver and peritoneal cavity, whereas SW480/miR-con cells only caused tumor increases without any metastasis (Figure 3C). [score:3]
However, less is known about the relationship between the miR-31 expression and the prognosis of patients with CRC. [score:3]
Xenograft tumors with miR-31-overexpression significantly formed the invasion of caecal wall (a,b), seeding metastasis of colon (c), stomach wall (d), abdominal wall (e) and diaphragm (f), and metastasis of lymph node (g), liver (h) and lung (i). [score:3]
The levels of miR-31 expression in CRC cells and tissues by qRT-PCR or in situ hybridization and its prognostic value in patients with CRC. [score:3]
Exogenetic over -expression of miR-31 promoted CRC cells growth, invasion, and migration in vitro. [score:3]
Ectopic expression of miR-31 decreased the endogenous levels of SATB2 mRNA and protein. [score:3]
Inhibition of miR-31 reduced the growth, invasion, and migration of CRC cells in vitro To confirm the effects of miR-31 on modulating the malignant phenotypes of CRC cells, we also investigated the change of aggressive phenotypes of CRC cells after reduced expression of miR-31. [score:3]
Over -expression of miR-31 was associated with an aggressive phenotype and poor prognosis of patients with CRC. [score:3]
We found that the over -expression of miR-31 resulted in significant reduction of SATB2 mRNA. [score:3]
Furthermore, the most important effect exerted by miR-31 on cell proliferation, invasion, and migration is partially reversed after transfection with a SATB2 expression vector. [score:3]
SATB2 can inhibit migration and invasion of SW480/miR-31. [score:3]
These results indicated that over -expression of miR-31 was sufficient to promote both cell proliferation and migration in vitro. [score:3]
In addition, it was revealed that miR-31 over -expression led to shorter overall survival times of the animals (p<0.05, Figure 3F). [score:3]
Furthermore, IHC staining illustrated that the tumors in caecum terminus of mice originating from SW480/miR-31 cells had increased expression of Snail and vimentin. [score:3]
SW480/miR-31 denoted CRC SW480 cells with stable over -expression miR-31. [score:3]
Correlation between the clinicopathological features and expression of miR-31. [score:3]
Furthermore, multivariate Cox regression analysis indicated that high-level expression of miR-31 is an independent prognostic factor for poor survival of patients with CRC (Table 2). [score:3]
Moreover, matrigel invasion and wound-healing assays confirmed that the inhibition of miR-31 expression reduced the invasiveness and migration of M5 and SW620 cells, compared to the control cells (p<0.05, Figure 4B and 4C). [score:3]
The previous studies have documented a number of deregulated miRNAs in CRC, including miR-21, miR-31 and so on. [score:2]
Matrigel invasion assay also illustrated that the over -expression of miR-31 markedly induced invasiveness of CRC cells (p<0.001 in both SW480 and DLD1, Figure 2G). [score:2]
In lung cancer cases, the knockdown of miR-31 substantially repressed lung cancer cell growth and carcinogenicity. [score:2]
Hence, miR-31 could be considered as a novel therapeutic target for patients with CRC. [score:2]
For patients with different levels of miR-31 expression, survival curves were plotted using the Kaplan–Meier method and compared using the log-rank test. [score:2]
But, the clinical prognostic significance, function and regulatory activity of miR-31 in CRC have not been completely understood yet. [score:2]
When compared with anti-miR-con transfection, an induced luciferase activity was shown in cells transfected with miR-31 inhibitor (p<0.001, Figure 5B). [score:2]
For tumor growth assay, a total of 2 × 10 [6] cells of SW480 with stable over -expression miR-31, or control cells, were injected subcutaneously in left and right flank of mice (n = 6 per group). [score:2]
An increasing number of in vitro studies have demonstrated an important role of miR-31 in regulating tumor growth, apoptosis, metastasis and chemotherapy resistance[14, 25– 27]. [score:2]
As shown in Figure 4A, when compared with the control cells, a significantly slower proliferation rate was observed in miR-31 inhibitor -transfected cells (p<0.001). [score:2]
Both miR-31 and SATB2 affect migration and invasion of CRC cells but in an opposite direction. [score:2]
Only the genes involved in metastasis cascade were considered as relevant targets with respect to the biological functions of miR-31 in CRC. [score:2]
We further compared the correlation between miR-31 and SATB2 expression. [score:2]
The results indicated that exogenetic expression of miR-31 in CRC cells caused a significant increase in cell migration using a wound-healing assay (p=0.001 in SW480, p<0.001 in DLD1, Figure 2F). [score:2]
Meanwhile, western blot assays showed that the protein levels of SATB2 were also substantially decreased after ectopic over -expression of miR-31 in SW480 and DLD-1 cells (Figure 5C and 5D). [score:2]
Thus, the miR-31/SATB2 pathway constitutes a previously unrecognized carcinogenesis and progression regulator of CRC. [score:2]
miR-31 facilitated CRC cells tumor growth and metastasis in vivo Since miR-31 promotes growth, migration, and invasion of CRC cells in vitro, we tempted to determine whether miR-31 could facilitate tumor growth and metastasis in vivo. [score:1]
As shown in Figure 3A, the speed of tumor growth in the SW480/miR-31 group was significant than that of the SW480/miR-con group (p<0.05, Figure 3A and 3B). [score:1]
In the presence of either miR-31 or miR-con, the firefly luciferase construct was co -transfected with a control Renilla luciferase vector pRL-CMV (Promega) into SW480 cells. [score:1]
The correlation analysis between clinicopathological characteristics and miR-31 level showed high-level expression of miR-31 was significantly associated with T-stage (p=0.045), lymph node metastasis (p=0.001), and distant metastasis (p=0.026) in patients with CRC, however, not associated with age, sex, tumor site, tumor size, and tumor differentiation degree (p>0.05, Table 1). [score:1]
The results indicated that, as an independent risk factor, miR-31 could serve as a prognostic marker for the survival of CRC patients. [score:1]
Cell growth rate (A) and colony formation (B) were measured to compare the growth difference between SW480/miR-31 cells and SW480/miR-31 cells with over -expression STAB2. [score:1]
In addition, miR-31 was low in SW480 and DLD-1 cells, which originate from primary tumors, whereas it was relatively high in SW620, Lovo, M5 and SCP51 cells, which originate from metastatic foci (Figure 1C), especially in M5 and SCP51 cell lines, which have high metastatic potential among the CRC cell lines. [score:1]
A stem-loop quantitative RT-PCR was carried out to detect expression of mature miR-31 with the ABI TaqMan [®]  MicroRNA Assay kit (Applied Biosystems, Foster City, USA) and gene-specific primers (Applied Biosystems, Foster City, USA) using an ABI 7500 Real-Time PCR system. [score:1]
Further investigations revealed that the over -expression of miR-31 in CRC led to increase tumor cell proliferation and motility in vitro and in vivo. [score:1]
To date, several studies have revealed the prognostic significance of miR-31 in various carcinomas, such as breast[14], bladder[31], and lung squamous cell carcinoma[32]. [score:1]
0085353.g005 Figure 5(A) Schematic illustration of the predicated miR-31 -binding sites (S1 and S2) in SATB2 3’-UTR. [score:1]
Flow cytometry and cell cycle analysis revealed a significant decrease in the percentage of cells in the G1/G0 phase (p=0.004 in SW480, p=0.008 in DLD1) and an increase in S phase of CRC cells treated with miR-31 (p=0.002 in SW480, p=0.001 in DLD1, Figure 2E). [score:1]
miR-31 promoted aggressive phenotypes of CRC cells in vitro. [score:1]
The sum of the staining-intensity and staining-extent scores was used as the final staining score for miR-31 (0–7). [score:1]
Since miR-31 promotes growth, migration, and invasion of CRC cells in vitro, we tempted to determine whether miR-31 could facilitate tumor growth and metastasis in vivo. [score:1]
In addition, 3’UTR of SATB2 mRNA contains two complementary sites for the binding region of miR-31. [score:1]
Staining for miR-31 was assessed using a relatively simple, reproducible scoring method[21, 22]. [score:1]
The contradictory results may indicate that miR-31 is tissue specific to a significant extent. [score:1]
The 3’UTR of SATB2 mRNA contained two complementary sites for the binding region of miR-31. [score:1]
miR-31 enhanced tumor growth, invasion and metastasis in vivo. [score:1]
However, Valastyan et al. illustrated the loss of miR-31 in metastatic breast cancer cell lines and patients. [score:1]
0085353.g006 Figure 6Cell growth rate (A) and colony formation (B) were measured to compare the growth difference between SW480/miR-31 cells and SW480/miR-31 cells with over -expression STAB2. [score:1]
Virus particles were harvested 48 hours after pLV-miR-31 transfection with the envelope plasmid pMD2. [score:1]
To the best of our knowledge, we presented the first large-scale study that combined in situ hybridization to evaluate the prognostic impact of miR-31 expression in CRC. [score:1]
However, miR-31 plays various functions in different cancers. [score:1]
We mutated several base pairs in the binding regions of the miR-31 binding site too (Figure 5A). [score:1]
Subsequently, 40 nM of a locked nucleic acid -modified, 5’ digoxigenin (DIG)-labeled oligonucleotide probe of hsa-miR-31 or a scrambled control probe (Exiqon) was added to the hybridization solution and hybridized at a temperature of 50°C overnight. [score:1]
Thus, the confirming results help us deducing that miR-31 is an oncogenic miRNA for CRC. [score:1]
The results indicated that miR-31 performed several functions as follows: it promoted cell growth and colony formation; it induced cell cycle G1/S transition of CRC cells in vitro; and it incited tumorigenesis in murine mo del of CRC xenograft. [score:1]
Body weight of control and SW480/miR-31 mice was also recorded every three days. [score:1]
This association indicated that miR-31 might well have a pivotal role in CRC metastasis. [score:1]
A 184-bp DNA fragment corresponding to pre-miR-31 was selected, and the flanking sequence was amplified and cloned into pLVTHM lentiviral vector (http://www. [score:1]
To confirm the effects of miR-31 on modulating the malignant phenotypes of CRC cells, we also investigated the change of aggressive phenotypes of CRC cells after reduced expression of miR-31. [score:1]
miR-31 could activate the RAS pathway and function as an oncogene in CRC by repressing RASA1 protein[27]. [score:1]
This association indicated that miR-31 might play a pivotal role in CRC metastasis. [score:1]
miR-31 facilitated CRC cells tumor growth and metastasis in vivo. [score:1]
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4
[+] score: 373
Our current data indicate that miR-31 directly inhibits expression of Dkk-1 and DACT3 in lung cancer cells as well as normal respiratory epithelia; furthermore, miR-31 over -expression in these cells depletes several other antagonists of Wnt signaling with potential miR-31 binding motifs including SFRP1, SFRP4, and WIF-1, which are frequently silenced in human lung cancers [53]. [score:8]
Whereas several reports have documented up-regulation of miR-31 in cultured lines or tissues derived from oropharyngeal, esophageal, lung, or colorectal carcinomas [45]– [48], our experiments appear to be the first to directly examine mechanisms regulating expression of this miRNA and its host gene in human lung cancer. [score:8]
Consistent with results pertaining to miR-31 (Figure 1A), 5-day CSC treatment induced expression of LOC554202, which was maintained for 20 days following removal of CSC from culture media (Figure 4A); these results were consistent with those pertaining to CSC -mediated up-regulation of miR-31 (Figure 1). [score:6]
The oncogenic effects of miR-31 in lung cancer cells were attributed to inhibition of large tumor suppressor 2 (LATS2) and PP2A regulatory subunit B alpha (PPP2R2A). [score:6]
Our findings, as well as those of Liu et al [47] are consistent with data demonstrating that up-regulation of miR-31 in oropharyngeal and colorectal carcinomas correlates with advanced stage of disease, and decreased survival of patients with these malignancies [45], [48]. [score:6]
F) of Dkk-1 and DACT3 expression in parental and vector control SAEC and Calu-6 cells, as well as SAEC and Calu-6 cells exhibiting constitutive over -expression or knock-down of miR-31. [score:6]
Over -expression of miR-31 significantly enhanced proliferation and tumorigenicity of lung cancer cells; knock-down of miR-31 inhibited growth of these cells. [score:6]
As shown in Figures 6A and B, constitutive over -expression of miR-31 increased proliferation of Calu-6 (35%) and H841(55%) relative to vector controls; in contrast, knock-down of endogenous miR-31 mediated ∼30% growth inhibition of these cancer cells. [score:6]
These experiments (Figure 1A) demonstrated that untreated Calu-6 and H841cells exhibited higher endogenous levels of miR-31 relative to SAEC and HBEC cells; additional analysis revealed that 5 day CSC exposure up-regulated miR-31 expression 5.5 fold and 3.6 fold in SAEC and HBEC, respectively relative to controls. [score:6]
Subsequent qRT-PCR experiments confirmed repression of SFRP1, SFRP4, and WIF-1, and up-regulation of Wnt-5a in SAEC and Calu-6 cells over -expressing miR-31 (Figure S1A). [score:6]
In addition, up-regulation of miR-31 mediates expression of the non-canonical ligand Wnt5a, which induces epithelial-to-mesenchymal transition (EMT), and dramatically enhances the malignant phenotype of lung cancer cells (Ripley et al, submitted for publication). [score:6]
Time course experiments revealed up-regulation of miR-31 in SAEC and H841 cells within 24 h following initiation of CSC exposure, with expression peaking and leveling off approximately 96 h later (Figure 1C). [score:6]
Consistent with data pertaining to up-regulation of miR-31 by CSC, expression of LOC554202 persisted for 20 days following cessation of CSC exposure. [score:6]
D) qRT-PCR analysis of Dkk-1 and DACT3 expression levels in SAEC, HBEC, Calu-6 and H841 cells with or without down-regulation of miR-31. [score:6]
CSC significantly increased miR-31 expression and activated LOC554202 in normal respiratory epithelia and lung cancer cells; miR-31 and LOC554202 expression persisted following discontinuation of CSC exposure. [score:5]
Herein, we report that CSC induces expression of miR-31 targeting several Wnt signaling antagonists including Dickkhopf-1 (Dkk-1) and DACT3 in normal human respiratory epithelia as well as lung cancer cells. [score:5]
qRT-PCR experiments revealed that 6 of 7 primary lung cancer specimens expressing ≥2 fold higher miR-31 levels relative to paired normal lung tissues also exhibited over -expression of LOC554202 (Figure 4B). [score:5]
Furthermore, over -expression of miR-31 diminished SFRP1, SFRP4, and WIF-1, and increased Wnt-5a expression. [score:5]
Reduction of miR-31 expression increased Dkk-1 as well as DACT3 expression in these cell lines (∼1.47–8 fold and ∼4.7–9 fold respectively; Figure 2D). [score:5]
B) qRT-PCR analysis of Dkk-1 and DACT3 expression levels in SAEC, HBEC, Calu-6, and H841 cells with or without over -expression of miR-31. [score:5]
Cigarette smoke induces expression of miR-31 targeting several antagonists of cancer stem cell signaling in normal respiratory epithelia and lung cancer cells. [score:5]
Over -expression of miR-31 significantly increased precipitation of Dkk-1 and DACT3 target transcripts in SAEC cells. [score:5]
Figure S1 A) qRT-PCR analysis demonstrating that over -expression of miR-31 decreases SFRP1, SFRP4 and WIF-1, and enhances Wnt5a expression in SAEC and Calu-6 cells. [score:5]
CLIP and reporter assays demonstrated direct interaction of miR-31 with Dickkopf-1 (Dkk-1) and DACT-3. Over -expression of miR-31 markedly diminished Dkk-1 and DACT3 expression levels in normal respiratory epithelia and lung cancer cells. [score:5]
Collectively, these experiments strongly suggested that miR-31 modulates Dkk-1 and DACT3 via post-transcriptional and translational -inhibitory mechanisms in cultured normal respiratory epithelia and lung cancer cells. [score:5]
Dkk-1 and DACT3 level were decreased, or somewhat enhanced in these cells following over -expression, or knock-down of miR-31, respectively. [score:4]
Dkk-1 and DACT3 are direct target candidates of miR-31. [score:4]
Shortly before this manuscript was initially submitted for publication, Liu et al [47] reported a series of experiments demonstrating up-regulation of miR-31 in lung cancer cells derived from cyclin E-transgenic mice. [score:4]
A/B) Direct cell count assays depicting in-vitro proliferation of Calu-6 and H841 lung cancer cells exhibiting over -expression or knock-down of miR-31. [score:4]
0013764.g006 Figure 6A/B) Direct cell count assays depicting in-vitro proliferation of Calu-6 and H841 lung cancer cells exhibiting over -expression or knock-down of miR-31. [score:4]
Knock-down of miR-31 increased Dkk-1 and DACT3 levels, and abrogated CSC -mediated decreases in Dkk-1 and DACT-3 expression. [score:4]
In contrast, knock-down of endogenous miR-31 appeared to increase Dkk-1 and DACT3 protein levels in SAEC and Calu-6 cells, although these changes did not correlate precisely with alterations in mRNA expression, possibly due, in part, to protein stability and antibody affinities. [score:4]
Interestingly, up-regulation of miR-31 has been observed in pulmonary adenomas or normal lung tissues from mice exposed to vinyl carbamate, or environmental smoke, respectively [42], [43]. [score:4]
Knock-down of miR-31 inhibited proliferation of mouse and human lung cancer cells, and diminished clonogenicity and tumorigenicity of murine lung cancer cells. [score:4]
Constitutive over -expression of miR-31 increased proliferation, whereas knock-down of miR-31 diminished proliferation of Calu-6 (a) as well as H841 (b) cells relative to vector controls. [score:4]
Consistent with these observations, qRT-PCR and ChIP experiments presented in this manuscript demonstrated that CSC -mediated up-regulation of miR-31 coincides with activation of LOC554202. [score:4]
Interestingly, up-regulation of miR-31 by CSC persisted for 20 days following removal of CSC from culture media (Figure 1B). [score:4]
Up-regulation of miR-31 in cultured cells exposed to CSC. [score:4]
Collectively, these data strongly suggest that C/EBP-β contributes to up-regulation of LOC554202 and miR-31 by CSC. [score:4]
WNT signaling pathway antagonists are direct target candidates of miR-31. [score:4]
A) Putative target sites of miR-31 within Dkk-1 3′ UTR (top) and DACT3 3′ UTR (bottom). [score:3]
RNA was isolated and purified from complexes, followed by PCR amplification of 3′ UTRs of miR-31 targets. [score:3]
miR-31 expression in primary lung cancer specimens. [score:3]
0013764.g002 Figure 2 A) miR-31 was over-expressed in SAEC, HBEC, Calu-6, and H841 cells via transient transfection of primary miR-31 constructs. [score:3]
Subsequent experiments revealed over -expression of miR-31 in human lung adenocarcinomas. [score:3]
Obvious increases in PCR products corresponding to 3′ UTRs of Dkk-1 and DACT3 were observed in SAEC over -expressing miR-31; this phenomenon was not observed in miR-31 -depleted SAEC cells. [score:3]
Subsequent experiments were undertaken to determine if depletion of endogenous miR-31 affected expression of Dkk-1 and DACT3 in cultured respiratory epithelial cells. [score:3]
Calu-6 and H358 cells constitutively over -expressing miR-31 and vector controls (1×10 [6]) were inoculated subcutaneously in the right and left flanks of athymic nude mice. [score:3]
Briefly, RNA cross-link immunoprecipitation (CLIP) techniques were used to detect interaction of miR-31 with these potential targets in SAEC cells [28]. [score:3]
As shown in Figures 6C and D, constitutive over -expression of miR-31 markedly increased volumes and masses of H358 as well as Calu-6 xenografts relative to control vectors (p<0.01). [score:3]
A) miR-31 was over-expressed in SAEC, HBEC, Calu-6, and H841 cells via transient transfection of primary miR-31 constructs. [score:3]
Expression of miR-31 in cultured cells exposed to CSC, primary lung cancers and adjacent normal lung tissues. [score:3]
β-actin served as the negative control since no sequences in the 3′ UTR that can be targeted by miR-31. [score:3]
Similar treatment increased miR-31 expression 3.1 and 6.5 fold in Calu-6 and H841 cells, respectively (Figure 1B). [score:3]
Subsequent analysis revealed over -expression of miR-31 in primary human lung cancer specimens –particularly those from smokers, relative to adjacent normal parenchyma. [score:3]
miR-31 and LOC554202 expression levels were significantly elevated in lung cancer specimens relative to adjacent normal lung tissues. [score:3]
qRT-PCR analysis confirmed high level miR-31 expression in miR-31 -transfected relative to control cells. [score:3]
experiments revealed ∼12 fold increases in miR-31 expression in miR-31 -transfected cells relative to vector controls (Figure 2A). [score:3]
Consistent with aforementioned results, western blot analysis revealed that Dkk-1 and DACT3 protein levels in SAEC and Calu-6 cells were decreased by over -expression of miR-31 (Figure 2F). [score:3]
As shown in Figure 1D, miR-31 expression was increased (mean 4.13 fold; range 1.47–12.46 fold) in lung cancers relative to paired normal lung tissues. [score:3]
Increased miR-31 expression was evident 20 days following discontinuation of CSC treatment. [score:3]
Volumes of xenografts derived from Calu-6 or H358 cells over -expressing miR-31 were significantly larger than vector controls (P<0.01). [score:3]
Over -expression of miR-31 decreased Dkk-1 as well as DACT3 (∼5–8 fold and ∼1.5–7 fold, respectively, relative to controls) in these four cell lines (Figures 2B). [score:3]
D) qRT-PCR analysis demonstrating miR-31 expression in human lung cancers relative to paired adjacent normal lung tissues. [score:3]
Software -guided analysis demonstrated that several antagonists of Wnt signaling including Dkk-1 [32] and DACT3 [33] were potential miR-31 targets. [score:3]
Collectively, these data confirmed preliminary experiments demonstrating higher levels of miR-31 expression in lung cancer cells relative to cultured normal respiratory epithelia (Figure 1A), and suggested that activation of miR-31 might be a biologically-relevant phenomenon during human pulmonary carcinogenesis. [score:3]
A) qRT-PCR analysis of endogenous miR-31 expression normalized with control miRNA (RNU44) in SAEC, HBEC, Calu-6, and H841cells. [score:3]
0013764.g003 Figure 3 A) Putative target sites of miR-31 within Dkk-1 3′ UTR (top) and DACT3 3′ UTR (bottom). [score:3]
Further studies are necessary to fully define the role of miR-31 in human malignancies, and specifically determine the prognostic and predictive significance of miR-31 expression during pulmonary carcinogenesis. [score:3]
Additional qRT-PCR experiments were performed to examine miR-31 expression levels in a randomly selected panel of primary non-small cell lung cancers and adjacent histologically normal lung parenchyma (clinical and pathologic data summarized in Table 1). [score:3]
In these experiments, β-actin served as the negative control since no sequences can be targeted by miR-31 (Figure 3B). [score:3]
B) qRT-PCR analysis of miR-31 expression in SAEC, HBEC, Calu-6, and H841cells cultured in normal media (NM) with or without CSC for 5 days. [score:3]
For miRNA target validation, approximately 2×10 [4] SAEC cells per well in 24-well plates were transiently transfected with 25 to 50 ng of each firefly luciferase reporter construct (Promega, Madison, WI), 150 to 175 ng pcDNA3 empty vector, 200-ng pRTK-Luc (Promega) as internal control, and 30 pmol of pre-miR-31 (SBI, Mountain View, CA). [score:3]
On the other hand, our data differ from those reported by Valastyan et al [56], Creighton et al [57], and Ivanov and colleagues [58], who have observed that miR-31 functions as a tumor suppressor in breast and ovarian carcinomas, as well as malignant pleural mesotheliomas. [score:3]
Of particular interest, aforementioned micro-array experiments suggested that CSC treatment induced expression of miR-31 in normal respiratory epithelia as well as lung cancer cells. [score:3]
As shown in Figure 2C, miR-31 expression levels were reduced ∼7–10 fold in SAEC, HBEC, Calu-6, and H841 cells transiently transfected with antisense-miR-31 (Zip-miR-31) constructs relative to vector controls. [score:3]
0013764.g001 Figure 1 A) qRT-PCR analysis of endogenous miR-31 expression normalized with control miRNA (RNU44) in SAEC, HBEC, Calu-6, and H841cells. [score:3]
Over -expression of miR-31 decreased Dkk-1 and DACT3 in all four cell lines. [score:3]
Additional experiments were undertaken to examine if miR-31 directly interacts with 3′ UTRs of Dkk-1 and DACT3. [score:2]
First, in-vitro growth assays were performed using Calu-6 and H841 cells exhibiting over -expression or depletion of miR-31. [score:2]
miR-31 negatively regulates WNT signaling pathway antagonists in normal respiratory epithelia and lung cancer cells. [score:2]
Collectively, these experiments strongly suggest that miR-31 directly interacts with 3′ UTRs of Dkk-1 and DACT3. [score:2]
Subsequent ChIP experiments demonstrated increased levels of H3K4Me3 and H3K9/14Ac activation marks in the proximal promoter region (0 to −1.5 k) of LOC554202 (which presumably contains the regulatory elements for miR-31) in SAEC following CSC exposure (Figures 4C and D). [score:2]
Knock-down of miR-31 enhances basal levels of Dkk-1 and DACT3 in these cells. [score:2]
MiR-31 expression significantly increased average mass of H358 as well as Calu-6 xenografts (p<0.001). [score:2]
Subsequent experiments revealed that knockdown of miR-31 significantly attenuated CSC -mediated decreases in Dkk-1 and DACT3 in SAEC as well as H841 cells (Figure 2E). [score:2]
The specific isoforms of C/EBP-β activating miR-31 and LOC554202, and the mechanisms mediating prolonged up-regulation of this miRNA and its host gene in respiratory epithelia and lung cancer cells following CSC exposure are a focus of ongoing investigation in our laboratory. [score:2]
Software -guided analysis, RNA cross-link immunoprecipitation (CLIP), 3′ UTR luciferase reporter assays, qRT-PCR, focused super-arrays and western blot techniques were used to identify and confirm targets of miR-31. [score:2]
E) qRT-PCR analysis demonstrating that knockdown of miR-31 partially blocks CSC -mediated decreases of Dkk-1 and DACT3 in SAEC. [score:2]
Collectively, these data suggest that miR-31 activates Wnt signaling in cultured lung cancer and normal respiratory epithelial cells. [score:1]
C) qRT-PCR analysis demonstrating time -dependent activation of miR-31 in SAEC and H841cells cultured in NM with or without CSC for 0, 12, 24, 48, 72, 96, and 120 hours. [score:1]
Additionally, Calu-6 as well as H358 cells stably -transfected with miR-31 or vector controls were inoculated subcutaneously in contralateral flanks of athymic nude mice (10 per group). [score:1]
Additional experiments were undertaken to ascertain if miR-31 enhances the malignant phenotype of lung cancer cells. [score:1]
To further investigate this phenomenon, quantitative RT-PCR (qRT-PCR) experiments were performed to examine miR-31 expression in SAEC, HBEC, Calu-6, and H841 cells cultured in normal media (NM) with or without CSC for 5 days. [score:1]
Our analysis revealed consistent activation of miR-31 in normal respiratory epithelia and lung cancer cells derived from smokers as well as never-smokers. [score:1]
Effects of miR-31 on proliferation and tumorigenicity of human lung cancer cells. [score:1]
miR-31 functions as an oncomir in lung cancer cells. [score:1]
These effects appeared somewhat more pronounced in normal SAEC and immortalized HBEC, possibly due to lower levels of endogenous miR-31 and higher levels of Dkk-1 and DACT3 in these cells relative to Calu-6 and H841 lung cancer cells. [score:1]
Recent ChIP analysis of distribution patterns of H3K4Me3, H3K9/14Ac, and H2AZ suggested that the host gene for miR-31 is LOC554202 [35]. [score:1]
Upstream sequencing of miRNAs by RNAPII ChIP-chip techniques revealed that LOC554202, which maps to 9p21.3, shares a TSS with miR-31, and acts as its host gene [35]. [score:1]
Super-arrays were used to further examine the effects of miR-31 on Wnt signaling in SAEC and Calu-6 cells. [score:1]
Role of C/EBP-β in CSC -mediated activation of miR-31. [score:1]
miR-31 functions as an oncomir during human pulmonary carcinogenesis. [score:1]
Our current data are consistent with, and extend these findings by demonstrating an additional potential mechanism by which miR-31 contributes to pulmonary carcinogenesis, namely activation of Wnt signaling. [score:1]
Interestingly, miR-31 levels in lung cancers from smokers were higher than those observed in non-smokers (5.2 vs 1.65 fold, respectively; p<0.01). [score:1]
Cell count and xenograft experiments were used to assess effects of miR-31 on proliferation and tumorigenicity of lung cancer cells. [score:1]
Collectively, these experiments strongly suggest that miR-31 enhances the malignant phenotype of human lung cancer cells. [score:1]
C) qRT-PCR analysis demonstrating decreased levels of endogenous miR-31 in SAEC, HBEC, Calu-6, and H841 cells following transient transfection of antisense-miR-31(Zip-miR-31) constructs relative to controls. [score:1]
Collectively, these observations suggest that miR-31 cooperates with epigenetic mechanisms to silence Wnt antagonists, thereby activating signaling networks implicated in maintenance of normal as well as cancer stem cells [54], [55] during initiation and progression of tobacco -induced lung cancers. [score:1]
Figure 3A depicts the potential miR-31 binding sites within the 3′ UTRs of Dkk-1 and DACT3 transcripts. [score:1]
Notably our analysis revealed a critical role for C/EBP-β in activation of miR-31 and LOC554202 by cigarette smoke. [score:1]
Effects of miR-31 on antagonists of WNT signaling. [score:1]
As such, additional experiments were performed to ascertain if this transcription factor contributed to CSC -mediated activation of miR-31 in cultured respiratory epithelia. [score:1]
Additional experiments were performed using pMiR-Report vectors with wt or mutant 3′ UTRs of Dkk-1 or DACT3, transiently co -transfected with miR-31 primary constructs or control vectors into SAEC. [score:1]
Epigenetic alterations coinciding with CSC -mediated activation of miR-31. [score:1]
To confirm these results, SAEC, HBEC, Calu-6, and H841 cells were transiently transfected with miR-31. [score:1]
C/EBP-β mediates CSC -induced miR-31 transcription. [score:1]
miR-31 levels in tumors were higher than corresponding normal lung. [score:1]
C) Schematic representation of LOC554202, the putative host gene of miR-31. [score:1]
B) CLIP analysis revealing interaction of miR31 with 3′ UTR of SFRP4. [score:1]
Consistent with aforementioned Wnt SuperArray results, additional CLIP experiments demonstrated interaction of miR-31 with SFRP4 (Figure S1B). [score:1]
Basal levels of miR-31 are higher in lung cancers relative to cultured normal or immortalized human respiratory epithelial cells. [score:1]
Discrepancies regarding our findings and those of the latter investigators may be attributable in part to genetic and epigenetic profiles of the various malignancies, reprogramming [59], tissue-specific effects of basal and inducible miR-31 expression, and Wnt signaling [60], [61]. [score:1]
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5
[+] score: 340
Other miRNAs from this paper: mmu-mir-125a, hsa-mir-125a, mmu-mir-31
miR-31 is universally expressed in a variety of tissues, and has been shown to negatively regulate lymphatic vascular lineage-specific differentiation 34, to sensitize breast cells to apoptosis by targeting protein kinase c-ɛ 35, to enhance vascular smooth muscle cell proliferation via inhibiting its target gene, the large tumor suppressor homologue 2 (ref. [score:12]
In contrast to a control construct lacking the target sequence, miR-31 overexpression led to significantly decreased luciferase activity derived from the construct expressing the target sequence, and a 88.12% reduction of the target ppp6c mRNA was achieved (Fig. 4i). [score:11]
Epidermal keratinocytes are responsive to dendritic cell-derived and T-cell-derived cytokines such as IFNs, TNF, IL-6, IL-17 and the IL-20 family of cytokines and vice versa, they are able to release proinflammatory cytokines and chemokines to sustain or even amplify the chronic inflammatory disease loop in lesional skin in psoriasis 1. Using the, the siRNA -mediated knockdown of NF-κB subunit p65 or luciferase reporter constructs driven by the specific promoter, we demonstrate that inflammatory cytokines are capable of triggering miR-31 transcription directly or indirectly through NF-κB signalling, which plays an essential role both in cell cycle regulation and inflammatory response, and critically connects keratinocytes with lymphocytes in the pathogenesis of psoriasis 4. Thus, the overexpression of miR-31 in psoriatic keratinocytes, likely as a consequence of the production of excess inflammatory cytokines in both skin lesion and plasma of patients, may therefore contribute to the epidermal hyperplasia that occurs in psoriasis. [score:9]
To identify putative target mRNAs of miR-31, four bioinformatics tools, TargetScan, miRDB, miRWalk and RNA22, were used to predict the potential targets of miR-31 (Supplementary Fig. 9a), and 20 potential target genes were identified (Supplementary Fig. 9b). [score:9]
Here we demonstrate that the conditional knockout of miR-31 leads to decreased keratinocyte hyperproliferation mediated by NF-κB signalling, prevents Ki67 expression, inhibits acanthosis and reduces the disease severity in two psoriasis mouse mo dels. [score:8]
Only one predicted target of miR-31, ppp6c, was significantly downregulated at the mRNA levels both after the application of IMQ and on the overexpression of miR-31 (Supplementary Fig. 9c). [score:8]
Fourth, miR-31 overexpression in NIH3T3 cells results in significantly decreased luciferase activity after the transfection of the cells with the construct expressing the target sequence in the 3′ UTR of ppp6c. [score:7]
The increased expression of miR-31 in diseased skin promoted us to assess its in vivo functionality in an imiquimod (IMQ) -induced psoriasis mouse mo del that closely resembles the human disease phenotype 23. [score:7]
Thus, our results demonstrate that miR-31 is capable of directly targeting a sequence within the 3′ UTR of the ppp6c mRNA and that ppp6c is one of the key targets of miR-31 in keratinocytes. [score:6]
We administered antagomirs to block the miR-31 seed sequence (anti-miR-31) and to test the inhibitory effects of anti-miR-31 on the disease development in the IMQ -induced psoriasis mouse mo del. [score:6]
Consistent with a published report 21, we showed that miR-31 expression was significantly upregulated in lesional skin from patients with psoriasis (Fig. 1f). [score:6]
A modest expression of ppp6c in the epidermis of the miR-31 [fl/fl] mice was observed in the absence of IMQ treatment; however, no apparent difference of ppp6c expression was found between the IMQ -treated and untreated c KO mice (Fig. 4a,b). [score:5]
Importantly, we observed that IL-6 expression was closely correlated with miR-31 expression in human psoriatic lesions (Fig. 2c and Supplementary Table 1). [score:5]
NF-κB signaling inhibits ppp6c expression by inducing miR-31. [score:5]
of ppp6c expression in NHEK with or without IL-6 treatment for 24 h. (h) Cell cycle analysis of primary mouse keratinocytes derived from miR-31 [fl/fl] or c KO mice in absence or presence of IL-6. Values were expressed as fold changes relative to non-stimulated HaCaT keratinocytes (e) or to non-stimulated NHEK (g) and normalized to β-actin. [score:5]
NF-κB signalling inhibits Ppp6c expression mediated by miR-31. [score:5]
Together, these experiments suggest that NF-κB activation triggered by inflammatory cytokines inhibits ppp6c expression mediated by miR-31 induction in keratinocytes. [score:5]
Thus, we suggest that IL-1α, IL-6, IL-17A, TNF-α, IFN-γ or IL-22 directly or indirectly activates the NF-κB signalling pathway mediating miR-31 expression in keratinocytes. [score:5]
Although activation of the STAT3 transcription factor is a classical downstream event of IL-6 stimulation, we found that there was no significant difference of the miR-31 levels in the HaCaT keratinocytes stimulated with IL-6 in the absence and presence of a STAT3 inhibitor (Supplementary Fig. 2b), indicating that the miR-31 expression induced by IL-6 may not be mediated by STAT3 signalling. [score:5]
miR-31 directly targets ppp6c, an essential element regulating cell cycle, which has been shown to be diminished in the epidermis of lesional skin from patients with psoriasis. [score:5]
Second, ppp6c expression is significantly decreased in diseased epidermis of miR-31 [fl/fl] control mice but not in the epidermis of c KO mice treated with IMQ or IL-23. [score:5]
Taken together, these data demonstrate that the expression of miR-31 is abundantly increased in the affected skin of psoriasis patients and mouse mo del, and support the notion that overexpressed miR-31 in hyperproliferating keratinocytes may be functionally involved in the pathogenesis of psoriasis. [score:5]
Although miR-31 was reported to modulate inflammatory cytokine and chemokine expression in keratinocytes by suppressing STK40 (ref. [score:5]
In contrast to the controls, the knockdown of p65 led to a significant decrease in miR-31 expression in keratinocytes stimulated with IL-6 (Fig. 2i). [score:4]
Here we identify that miR-31 induced by NF-κB activation directly targets ppp6c to promote keratinocyte hyperproliferation. [score:4]
In our study NF-κB acts as an upstream enhancer of miR-31 in keratinocytes, while another report shows that miR-31 targets serine/threonine-protein kinase 40 (STK40), a negative regulator of the NF-κB signalling pathway 21. [score:4]
Our data identify miR-31 as a downstream target of NF-κB and highlight the critical role of NF-κB -mediated post-transcriptional regulation for epidermal hyperplasia in psoriasis. [score:4]
We next investigated the role of miR-31 in the development of IMQ -induced psoriasiform skin disease, and found that the miR-31 [TG] mice displayed a more severe form of the disease than the control mice (Supplementary Fig. 4a–d). [score:4]
We compared the mRNA expression profile of the predicted targets in the WT and miR-31 [TG] mice. [score:4]
miR-31 directly targets ppp6c. [score:4]
miR-31 directly targets Ppp6c. [score:4]
The data presented here show that NF-κB induces miR-31, which directly inhibits ppp6c, thereby increasing basal keratinocyte proliferation. [score:4]
Expression of miR-31 in lesional skin of psoriasis mouse mo dels and patients. [score:3]
Almost all of the tested inflammatory cytokines were able to stimulate miR-31 expression to different extents; however, IL-6 appeared to be an inducer of miR-31 at low concentrations in NHEK (Fig. 2b). [score:3]
To create loxP-miR-31-loxP mice, a targeting vector was designed to insert with an frt-flanked PGK-neo cassette and a loxP site upstream of miR-31 and a second loxP site downstream of miR-31. [score:3]
These findings indicate that miR-31 and its target gene ppp6c are specifically associated with Ago2-containing complexes. [score:3]
Two out of five founders were selected for further breeding and experiments according to the expression levels of miR-31 in peripheral blood mononuclear cells. [score:3]
The increased ppp6c expression after the anti-miR-31 treatment in epidermis was further confirmed by immunohistochemistry analysis (Fig. 7f). [score:3]
To further confirm whether IL-1α, IL-6, IL-17A, TNF-α, IFN-γ and IL-22 are able to induce the expression of miR-31 through NF-κB signalling, we used luciferase reporter constructs driven by miR-31-specific promoter response in HaCaT keratinocytes. [score:3]
Thus, our data again indicate that miR-31 and its target ppp6c are critical factors in epidermal hyperplasia in psoriasis. [score:3]
In conclusion, the present study has shown that the activation of NF-κB signalling triggered by inflammatory cytokines induces miR-31 expression in keratinocytes. [score:3]
NF-κB activation induces miR-31 expression. [score:3]
Of the 610 miRNAs analysed, we observed that miR-31 was the miRNA that was most highly expressed in affected skin (4.3-fold increase) (Fig. 1a,b). [score:3]
We present several lines of evidence to support that ppp6c is one of the primary targets for miR-31 in keratinocytes. [score:3]
miR-31 expression is elevated in psoriatic skin. [score:3]
Ppp6c inhibition is required for miR-31 -mediated effects. [score:3]
Notably, by quantitative real-time PCR (qPCR), we determined that miR-31 was significantly increased in the lesional skin from IMQ -treated mice compared with the healthy skin (1±0.2276 versus 3.877±0.4426, n=13, P<0.0001, two-tailed Student's t-test) (Fig. 1c), and the upregulated miR-31 was mainly confined to epidermis (Fig. 1d). [score:3]
Furthermore, we observed that the miR-31 deletion in the mice treated with IMQ significantly restored the expression of the terminal differentiation markers Keratin 10, Loricrin and Filaggrin close to that of the untreated miR-31 [fl/fl] mice (Fig. 3m). [score:3]
Moreover, the deletion of miR-31 in keratinocytes did not decrease miR-31 expression in splenocytes in the c KO mice treated with IMQ (Supplementary Fig. 8b). [score:3]
First, four separate bioinformatics tools predict that miR-31 targets a sequence in the 3′ UTR of ppp6c mRNA. [score:3]
We found that miR-31 expression was restricted to the basal and suprabasal cell layers of the epidermis in the lesional skin of the IMQ -treated mice (Fig. 1e). [score:3]
Purple colour indicates miR-31 expression. [score:3]
Furthermore, we determined that ppp6c and miR-31 expression in the miR-31 [fl/fl] epidermal immunoprecipitates were 5.0- and 9.1-fold greater, respectively, than those in the c KO mice (Fig. 4e,f). [score:3]
Unlike ppp6c there was essentially no difference of the expression levels for a non-related miRNA miR-125a in the psoriatic epidermal immunoprecipitates between miR-31 [fl/fl] and c KO mice treated with IMQ (Supplementary Fig. 11a,b). [score:3]
miR-31 conditional deletion reduces the disease severity. [score:3]
Third, ppp6c and miR-31 expression in Ago2 immunoprecipitates of lesional epidermis derived from miR-31 [fl/fl] animals is markedly enriched compared with that in immunoprecipitates from c KO mice, suggesting that ppp6c and miR-31 associate within Ago2, the effector element of the miRNA -induced silencing complex, which directly binds to miRNAs and subsequently mediates mRNA repression 39. [score:3]
Inhibition of ppp6c is functionally important for the biological effects of miR-31 in epidermal hyperplasia. [score:3]
These data indicate that ppp6c is a potential miR-31 target. [score:3]
Moreover, we detected that NHEK exhibited an enhanced proliferation after overexpressing miR-31 (Supplementary Fig. 5). [score:3]
However, silencing ppp6c did not alter the miR-31 expression in NHEK (Supplementary Fig. 11c). [score:3]
To confirm whether ppp6c is a direct target of miR-31, Ago2 immunoprecipitates of the epidermis derived from untreated and IMQ -treated mice or from miR-31 [fl/fl] and c KO mice treated with IMQ were assayed for ppp6c and miR-31. [score:3]
These findings that suggest different target genes of miR-31 in fact indicate that the miR-31 -mediated positive feedback loop may amplify NF-κB activity to pathological levels in epidermal hyperplasia. [score:3]
21), the in vivo function of miR-31 and the underlying mechanism by which it regulates cell proliferation and differentiation in psoriasis has been poorly explored. [score:2]
Clearly, miR-31 expression was significantly decreased in the psoriatic lesions in the IL-17A [−/−] mice compared with the IL-17A [+/+] mice after the IMQ application (Fig. 2a). [score:2]
To our knowledge, no prior studies have addressed the possible direct intrinsic role of miR-31 in keratinocyte proliferation or differentiation and in the pathogenesis of psoriasis. [score:2]
We sought to investigate whether the upregulation of Th17 cytokines coincides with the induction of miR-31 in skin inflammation. [score:2]
Notably, we detected that the expression of Ki67, a marker strictly associated with cell proliferation, was significantly decreased in the c KO mice when compared with the controls, indicating that the excessive proliferation of basal keratinocytes induced by IMQ was reduced in the absence of miR-31 (Fig. 3k,l). [score:2]
In this study, we demonstrate that inflammatory cytokines activate NF-κB signalling and induce miR-31, which represses ppp6c, a negative regulator of the cell cycle, thereby contributing to basal keratinocyte proliferation and epidermal hyperplasia. [score:2]
Consistent with our qPCR results, western blot analysis further demonstrated that the ppp6c expression was increased by >3.7-fold in the epidermis of the c KO mice treated with IMQ compared with that of miR-31 [fl/fl] controls. [score:2]
The germline-transmitted mice were further crossed with Keratin 5-Cre transgenic mice to achieve a conditional knockout mouse mo del with a deleted miR-31 allele in the epidermal basal keratinocytes (Fig. 3b–d). [score:2]
We observed that the conditional knockout of miR-31 in basal keratinocytes dramatically decreased splenomegaly and lymphadenopathy in the IMQ -treated mice (Fig. 3f). [score:2]
In addition, miR-31 expression in epidermis even decreased in the c KO mice treated with IMQ compared with the untreated c KO controls (Supplementary Fig. 8a), indicating that infiltrated leucocytes did not contribute to the elevated levels of miR-31 in the epidermis. [score:2]
In an IL-23 -mediated mouse mo del of psoriasis, the ppp6c expression was increased more than ∼8.0-fold at the mRNA levels and 12.0-fold at the protein levels in the ear epidermis of the c KO mice injected with IL-23 compared with that of the miR-31 [fl/fl] controls (Supplementary Fig. 10a,b). [score:2]
We found that IL-1α, IL-6, IL-17A, TNF-α, IFN-γ and IL-22 stimulation increased p65 binding to the putative binding site at −130 of the putative promoter of miR-31, and mutation of the binding site at −130 blocked the luciferase activity induced by these cytokines (Fig. 2j). [score:2]
Cells with 60% confluence were co -transfected with 100 ng 3′-UTR luciferase reporter vector and 50 pmol miR-31 mimics (GenePharma) using TurboFect (Thermo Scientific, #R0531). [score:1]
were injected subcutaneously with an irrelevant antagomir (NC) or an antagomir to miR-31 (anti-miR-31). [score:1]
Decreased epidermal hyperplasia and dermal cellular infiltrates in miR-31 [fl/fl]/K5-Cre mice treated with IMQ. [score:1]
html) is on chromosome 4 (Mus musculus) and encodes the miR-31. [score:1]
To confirm this observation, we performed in situ hybridization on skin cryosections from the IMQ -treated mice using miR-31-specific locked nucleic acid -modified (LNA) probes. [score:1]
The resulting loxP-miR-31-loxP mice were backcrossed onto C57BL/6J background for five generations and bred with Keratin 5-Cre transgenic mice. [score:1]
The conditional deletion of miR-31 decreases epidermal hyperplasia and attenuates psoriasiform phenotype in mouse mo dels of psoriasis. [score:1]
Generation of miR-31 [fl/fl]/Keratin 5-Cre mice. [score:1]
Generation of miR-31 [fl/fl]/Keratin 5-Cre miceThe miR-31 locus (mmu-mir31 ENSMUSG00000065408, http://www. [score:1]
In vivo miR-31 interference AntagomiR-31 with sequences complementary to mature miR-31, complete 2′-O-methylation of sugar, phosphorothioate backbone and a cholesterol-moiety at 3′-end were synthesized by RIBOBIO (Guangzhou, China). [score:1]
The miR-31 locus (mmu-mir31 ENSMUSG00000065408, http://www. [score:1]
P1 and P2 were used to genotype the miR-31 floxed allele (1,064 bp) and the miR-31 deleted allele (235 bp). [score:1]
Consistently, we found that miR-31 deletion in the epidermis led to an obvious decrease in plaque formation accompanied with a significant decrease in ear thickness and acanthosis in an IL-23 -mediated psoriasis mouse mo del (Supplementary Fig. 6a–d). [score:1]
Briefly, after incubated in acetylation solution (0.02 M HCl, 1.3% trietanolamin and 0.25% acetic anhydride in diethyl pyrocarbonate -treated water) for 10 min at room temperature (RT), sections were treated with proteinase K (5 μg ml [−1]) in PBS for 10 min, washed and prehybridized for 6 h. Hybridization with mmu-miR-31 5′-DIG and 3′-DIG -labelled miRCURY LNA detection probe (Exiqon, #39153-15) was performed overnight at 50 °C. [score:1]
The critical role of miR-31 in hyperproliferative keratinocytes is further indicated by the observation that repeated intradermal (i. d. ) injection of anti-miR-31 in the IMQ -induced mouse mo del results in a significant improvement of the psoriasiform phenotype. [score:1]
Fifth, the administration of anti-miR-31 blocks miR-31 function, and enhances the ppp6c mRNA and protein levels in vivo. [score:1]
Error bars depict s. e. m. How to cite this article: Yan, S. et al. NF-κB -induced microRNA-31 promotes epidermal hyperplasia by repressing protein phosphatase 6 in psoriasis. [score:1]
As expected, there was a pronounced decrease in both acanthosis and dermal cellular infiltration after the anti-miR-31 treatment (Fig. 7a–c). [score:1]
The levels of ppp6c and miR-31 detected in the psoriatic epidermal immunoprecipitates were 11.2- and 49.1-fold greater, respectively, than those in untreated controls (Fig. 4c,d). [score:1]
Generation of miR-31 [TG] mice. [score:1]
Correspondingly, we found enhanced ppp6c mRNA and protein levels in the epidermis after the administration of anti-miR-31 (Fig. 7d,e). [score:1]
Together, these data imply that intrinsic miR-31 plays a pivotal role in keratinocyte proliferation and differentiation, and in epidermal hyperplasia. [score:1]
To further study the potential role of miR-31 in the aetiology and pathogenesis of psoriasis, we created miR-31 transgenic mice (miR-31 [TG]) using a viral vector carrying enhanced green fluorescent protein (EGFP) and pri-miR-31 under the control of the cytomegalovirus (CMV) promoter (Supplementary Fig. 3a,b). [score:1]
AntagomiR-31 with sequences complementary to mature miR-31, complete 2′-O-methylation of sugar, phosphorothioate backbone and a cholesterol-moiety at 3′-end were synthesized by RIBOBIO (Guangzhou, China). [score:1]
miR-31 [TG] mice did not develop any skin phenotypes at basal conditions on either strain of mice at least for 6 months. [score:1]
Moreover, the anti-miR-31 administration markedly reduced the keratinocyte hyperproliferation as indicated by the Ki67 levels (Fig. 7g). [score:1]
miR-31 [fl/fl] and c KO mice did not develop any skin phenotypes at basal conditions on either strain of mice at least for 6 months. [score:1]
Thus, we chose IL-6 as a stimulus for miR-31 induction. [score:1]
We then applied IMQ to both the miR-31 control (miR-31 [fl/fl]) and c KO mice, and found that miR-31 deficiency led to a pronounced decrease in plaque formation (Fig. 3e). [score:1]
Strikingly, we demonstrated that the miR-31-specific ablation in the epidermis resulted in a pronounced decrease in skin thickness in the c KO mice treated with IMQ (Fig. 3g). [score:1]
Simple linear regression mo del was used to analyse the correlation between RNA levels of IL-6 and miR-31. [score:1]
Of note, we detected a dramatic increase of proliferation in the miR-31 [fl/fl] keratinocytes, but not the c KO keratinocytes when stimulated with IL-6, indicating that IL-6 is not able to trigger keratinocyte proliferation in the absence of miR-31 (Fig. 6h). [score:1]
We sought to investigate whether IL-6 could trigger the expression of miR-31 mediated by NF-κB signalling in keratinocytes. [score:1]
However, in addition to IL-1α there was no significant difference of the inflammatory genes between the miR-31 [fl/fl] and c KO mice in both the IMQ -induced and IL-23 -mediated mouse mo dels of psoriasis (Supplementary Fig. 7), suggesting a unique role of miR-31 in epidermal hyperplasia. [score:1]
A database analysis identified one potential p65 -binding site in the promoter element at −130 upstream from the transcription start site of human miR-31 (Fig. 2f). [score:1]
The skin of the miR-31 [fl/fl]/Keratin 5-Cre (c KO) mice remained healthy without any detectable inflammatory pathology for at least 32 weeks. [score:1]
Requirement of NF-κB signaling for the induction of miR-31 in epidermal keratinocytes. [score:1]
To examine the possible involvement of key inflammatory cytokines in the induction of miR-31, we stimulated primary normal human epidermal keratinocytes (NHEK) with IL-1α, IL-6, IL-17A, IL-22, interferon-γ (IFN-γ) and TNF-α. [score:1]
In vivo miR-31 interference. [score:1]
The complementary DNAs were then analysed by qPCR using the TaqMan probes for miR-31 and U6 snRNA. [score:1]
Anti-miR-31 administration decreases epidermal hyperplasia. [score:1]
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Other miRNAs from this paper: hsa-mir-93, hsa-mir-198, hsa-mir-181a-2, hsa-mir-191
Given that miR-31 was the only common target identified from these two approaches (downregulated in EMSY amplified cases and upregulated in cells depleted from EMSY) and that miR-31 has been previously reported as a microRNA involved in the suppression of metastasis in breast cancer (Valastyan et al., 2009a, 2009b, 2010, 2011; Valastyan and Weinberg, 2010, 2011), we considered the possibility that miR-31 is an EMSY target gene and that it may be part of the mechanism underpinning EMSY’s oncogenic potential. [score:12]
In breast cancer cell lines, the overexpression of EMSY reduces the expression of the miR-31 gene, increases the expression of miR-31 target genes, and induces invasion and migration. [score:9]
In order to examine the relationship between EMSY amplification, miR-31 expression levels, and the expression of miR-31 targets, we employed the METABRIC cohort of ∼2,000 primary breast tumors for which paired Affymetrix SNP 6.0 copy-number data and Illumina HT-12 expression data were available. [score:9]
Given that miR-31 affects cell invasion and migration via its pleiotropic regulation of prometastatic target genes (Valastyan et al., 2009b), we tested whether EMSY expression impacts on miR-31 target genes. [score:8]
To test this, we analyzed MCF-7 cells stably overexpressing EMSY (Figures 1B and S1B) for the expression of a set of validated miR-31 target genes (Valastyan et al., 2009a, 2009b). [score:7]
These findings are consistent with the results obtained from the mammary fat pad and tail vein injection experiments described in Figure 1. Then, to establish whether miR-31 mediates, at least in part, the effects of EMSY overexpression, we performed a “rescue” experiment wherein miR-31 was exogenously expressed in the context of EMSY overexpression. [score:7]
Figures 5B and S5A show that, of the three TFs, only ETS-1 downregulation led to the specific upregulation of miR-31. [score:7]
We also find that loss of miR-31 did not enhance transformation in MCF-7 cells, suggesting a two-step process, wherein EMSY overexpression induces cell transformation and then EMSY functions to downregulate miR-31, leading to the progression of the transformed phenotype (i. e., the acquisition of traits such as invasion and migration). [score:6]
These data indicate that EMSY overexpression induces MCF-7 cell transformation, and, in a second step, EMSY functions to downregulate miR-31, leading to the progression of the transformed phenotype (i. e., the acquisition of traits such as invasion and migration). [score:6]
This analysis revealed that miR-31 levels were significantly reduced in cells stably overexpressing EMSY, whereas the expression of other miRNAs such as miR-181a-2 and miR-198 remained unchanged (Figure 2C). [score:5]
Given that the expression probe for EMSY on the Illumina HT12 v3 array was nonresponsive, we profiled miR-31 expression levels by qRT-PCR in a representative subset of 98 primary tumors from the METABRIC cohort. [score:5]
miR-31 expression levels were binned into low and high states on the basis of the lower and upper 15% of expression values, respectively. [score:5]
EMSY is not listed as a putative miR-31 target, and its expression remained constant upon miR-31 transfection (data not shown). [score:5]
The expression of miR-31 was monitored by qRT-PCR in MCF-7 cells overexpressing EMSY and in comparison to control cells. [score:5]
Moreover, restoration of miR-31 significantly inhibited the invasion, migration, and colony-formation abilities of cells overexpressing EMSY or harboring EMSY amplification, a result which phenocopied the effects of EMSY depletion in these cells. [score:5]
We found that EMSY expression strongly anticorrelated with miR-31 expression (ρ = −0.732, Spearmann’s rho; p value < 0.0001; Figures S2B and S2C). [score:5]
Re -expression of miR-31 profoundly reduced cell migration, invasion, and colony-formation abilities of cells overexpressing EMSY or haboring EMSY amplification. [score:5]
Moreover, increased expression of miR-31 phenocopies the effect of EMSY knockdown in these two EMSY-amplified cell lines (Figures 3G and S3E). [score:4]
The depletion of BRCA2 has no effect on miR-31 expression, indicating that BRCA2 is not directly involved in the EMSY/miR-31 pathway (Figure S4C). [score:4]
Conversly, and consistent with these findings, miR-31 was markedly upregulated in EMSY -depleted MCF-7 cells (Figure S2E), in comparison to miR-181a-2 and miR-198 (Figure 2D). [score:4]
Then, we asked whether the re -expression of miR-31 in MCF-7 cells stably overexpressing EMSY could abrogate EMSY -induced oncogenic activity using the soft-agar assay. [score:4]
Then, qRT-PCR was performed in order to assay EMSY and miR-31 expression levels as well as the expression of the control miRNAs, miR-191, and miR-93. [score:4]
Figure 3E shows that miR-31 was markedly upregulated in MDA-MB-175 cells upon EMSY depletion, consistent with our results in MCF-7 cells (Figure 2C). [score:4]
For example, in adult T cell leukemia, Polycomb proteins have been shown to contribute to miR-31 downregulation (Yamagishi et al., 2012). [score:4]
Notably, downregulation of EMSY does not affect the binding of ETS-1 to the miR-31 promoter, supporting a mo del in which ETS-1 recruits EMSY to the miR-31 promoter. [score:4]
Furthermore, we found that RNA polymerase II occupancy on the miR-31 promoter increased upon the downregulation of EMSY, consistent with EMSY repressing transcription (Figure 4C). [score:4]
Indeed, recent reports have identified other genetic and epigenetic axes influencing miR-31 expression (Augoff et al., 2012; Yamagishi et al., 2012). [score:3]
Altogether, these results support an inverse correlation between EMSY and miR-31 expression levels. [score:3]
Given that miR-31 has been described to affect invasion and migration (Valastyan et al., 2009b, 2010), we tested whether miR-31 inhibition alters the initial acquisition of a transformed phenotype. [score:3]
In order to validate the TF binding site predicions, we depleted MCF-7 cells for each of the TFs with a putative binding site in the miR-31 promoter (ETS-1, ETV4, and GATA1) and assessed the expression of miR-31. [score:3]
Figure 3D shows that miR-31 re -expression profoundly reduces the ability of the MCF-7-EMSY cells to form colonies in soft agar. [score:3]
Moreover, miR-31 is a critical target of EMSY in breast cancer. [score:3]
Exogenous expression of miR-31 reduced the invasive and migrative rates of both cells lines (Figures 3H and S3F). [score:3]
In addition, another epigenetic modification, DNA methylation, is also involved in decreasing miR-31 expression in breast cancer (Vrba et al., 2013). [score:3]
Examination of the large METABRIC cohort highlights a negative correlation between EMSY and miR-31 expression. [score:3]
This effect is specific, given that non-miR-31 target transcripts, namely B2M, CXCL12, and ALAS1, remain unaffected (Figure 2E). [score:3]
Enforced expression of miR-31 significantly reversed EMSY -mediated induction of cell migration (Figure 3A). [score:3]
MCF-7 cells stably overexpressing EMSY were transfected with a vector encoding miR-31. [score:3]
Three miR-31 target genes, namely ITGA5, RDX, and RhoA, are crucial for the antimetastatic response of miR-31 (Valastyan et al. 2009a, 2009b). [score:3]
Therefore, we conclude that miR-31 expression phenocopies the effect of EMSY depletion. [score:3]
Finally, we monitored the invasion and migration abilities of MDA-MB-175 and MDB-MB-415 cells after the depletion of EMSY or overexpression of miR-31. [score:3]
EMSY is recruited to the miR-31 promoter by the ETS-1 TF, and, together with the KDM5B histone demethylase, these factors repress miR-31 expression (Figure 5). [score:3]
Next, we set out to further confirm the observation that miR-31 expression levels are significantly lower in EMSY-amplified versus neutral tumors (Figure 2B; p value < 0.001; Wilcoxon rank-sum test). [score:3]
Importantly, it did so without affecting the expression of miR-198 and miR-181a-2. To further confirm these results and validate the predictions from the bioinformatic analyses, we tested the ETS-1 motifs in the miR-31 cis-regulatory element in functional transcription assays. [score:3]
EMSY’s contribution to tumorigenesis may not be solely due to the silencing of miR-31, given that other EMSY unidentified target genes may also play a role. [score:3]
Figure S2D shows that miR-31 expression is lower in tumors generated from the MCF-7-EMSY than from MCF-7 control cells. [score:3]
We identify an inverse correlation between EMSY amplification and miR-31 expression, an antimetastatic microRNA, in the METABRIC cohort of human breast samples. [score:3]
These results demonstrate that each of the ETS-1 binding sites are relevant to miR-31 expression and that ETS-1 functions as a repressor in this context. [score:3]
Consistent with its role as a histone demethylase, the depletion of KDM5B (Figure S5D) resulted in an increase of H3K4 trimethylation on the miR-31 promoter (Figure 5F) and a concomitant increase in the expression of miR-31 (Figure 5G). [score:3]
• EMSY is an oncogene that represses miR-31 transcription in a BRCA2-independent manner • Re -expression of miR-31 abrogates EMSY -mediated effects on cell migration in vitro • EMSY and miR-31 levels anticorrelate in human breast samples from the METABRIC cohort • EMSY, ETS-1, and KDM5B co-occupy miR-31 promoter and repress its transcription The amplification of EMSY has been found in 17% and 13% of sporadic ovarian and breast cancers, respectively, and it is associated with a poor outcome (Brown et al., 2006, 2008, 2010; Hughes-Davies et al., 2003; Raouf et al., 2005; Rodriguez et al., 2004). [score:3]
Then, we further examined the relationship between EMSY and miR-31 expression levels. [score:3]
Altogether, these observations indicate that EMSY associates with the promoter of miR-31 and represses its expression. [score:3]
Moreover, the EMSY/ETS-1/KDM5B pathway may not be the only route for miR-31 transcriptional regulation. [score:2]
Thus, the regulation of the miR-31 pathway by EMSY can explain, at least in part, its oncogenic behavior and association with poor prognosis (Figure 3). [score:2]
Importantly, we confirmed by ChIP that the ETS-1 protein binds directly to the miR-31 promoter within the region containing the predicted ETS binding site (Figure 5D). [score:2]
The mutation of both ETS-1 binding sites (218 and 512) did affect miR-31 promoter activity more than each of the single mutants. [score:2]
Figure 5A shows that putative binding sites for ETS family members (ETS-1 and ETV4/PEA3) can be found in the miR-31 regulatory region but not within the analogous regions of miR-198 and miR-181a-2, whereas a GATA1 site is present within miR-31 and miR-198. [score:2]
One of them, miR-31, is a key regulator of breast cancer metastasis. [score:2]
Altogether, these results support a mo del whereby ETS-1 directly binds to an ETS binding motif within the miR-31 promoter to recruit EMSY and KDM5B to repress transcription. [score:2]
We used two such cell lines, MDA-MB-175 and MDA-MB-415 (Rodriguez et al., 2004), and confirmed that both have high EMSY and very low miR-31 levels in comparison to MCF-7 cells (Figure S3A). [score:1]
The Transcription Factor ETS-1 Recruits EMSY to the miR-31 Promoter. [score:1]
The pathway indentified here (EMSY/ETS1/KDM5B/miR-31) may not provide the unique and complete molecular explanation for the association between EMSY amplification and poor prognosis. [score:1]
We found that a loss of miR-31 significantly enhanced the invasion and migration capacity of MCF-7 cells (Figure 3B). [score:1]
Depletion of KDM5B did not affect the association of either EMSY or ETS-1 to the miR-31 promoter but did affect its own binding in that region (Figure 5F). [score:1]
We identified the promoter of miR-31 using 5′ rapid amplification of complementary DNA (cDNA) ends (RACE) experiments (Figure S4A). [score:1]
miR-31 Reverts EMSY Oncogenicity In Vitro. [score:1]
Importantly, these data are completely consistent with those reported by Valastyan et al. (2009b) and led us to propose a mo del for the EMSY-miR-31 interaction. [score:1]
Human miR-31 promoter was amplified by PCR from genomic DNA of MCF-7 cells. [score:1]
Figure 5C shows promoter activity for the miR-31 promoter region containing the 2 ETS-1 motifs. [score:1]
Moreover, ChIP experiments showed that KDM5B binds to the miR-31 promoter at the same position as EMSY and ETS-1 (Figures 5F and S5E). [score:1]
ChIP analyses indicated that EMSY associated with the promoter of miR-31 but did not bind the regions upstream of two control miRNA genes, miR-181a-2 and miR-198 (Figures 4B and S4B). [score:1]
This analysis also showed that ETS-1 did not bind to other regions within the miR-31 promoter, nor did it bind to the promoters of miR-181a-2 or miR-198 (Figure S5C). [score:1]
Thus, the loss of miR-31 from MCF-7 cells did not enhance transformation. [score:1]
In contrast, the depletion of EMSY led to increased levels of primary miR-31 transcripts (pri-miR-31; Figure 4A) suggesting that EMSY affects the transcription of the miR-31 gene rather than the processing of its transcripts. [score:1]
Cases were also selected to be copy-number neutral for IFNE1 and MTAP, which flank the miR-31 locus. [score:1]
EMSY occupancy on the miR-31 promoter was higher in cells with EMSY amplification, whereas RNA polymerase II occupancy was lower (Figure 4D). [score:1]
We used antisense oligonucleotides to deplete MCF-7 cells from miR-31. [score:1]
EMSY Represses miR-31 Transcription and Binds to the miR-31 Promoter. [score:1]
Then, we examined the DNA sequence upstream of the miR-31 transcription start site for TF binding sites. [score:1]
To further understand the mechanisms by which EMSY silences miR-31, we sought to decipher how EMSY is recruited to the miR-31 promoter. [score:1]
However, when miR-31 sponges were used to stably deplete miR-31 from MCF-7 cells, we observed that the resulting cells formed colonies in soft agar as efficiently as control MCF-7 cells (Figure 3C). [score:1]
Figure 2E shows that MCF-7-EMSY cells display higher levels of transcripts reported to be under the control of miR-31 (RhoA, RDX, ITGA5, M-RIP, FZD3, and MMP16) in comparison to control cells. [score:1]
Using these data, we evaluated the relationship between EMSY amplification and miR-31 expression levels as well as the association between these events and clinical outcome. [score:1]
Given the convergence of four breast-cancer -associated genes (EMSY, ETS-1, KDM5B, and miR-31) with overlapping biological roles, this pathway offers a number of avenues for therapeutic intervention. [score:1]
Chromatin immunoprecipitation (ChIP) experiments show that EMSY binds to the miR-31 promoter. [score:1]
Furthermore, depletion of ETS-1 resulted in reduced binding of both ETS-1 and EMSY to the miR-31 promoter (Figure 5D) without affecting the levels of EMSY protein in MCF-7 cells (Figure S5B). [score:1]
We constructed reporter vectors containing the miR-31 promoter sequence upstream of the firefly luciferase cDNA using the pGL4 plasmid. [score:1]
The Histone H3K4me3 Demethylase KDM5b Interacts with EMSY and Contributes to miR-31 Silencing. [score:1]
We show that EMSY is recruited to the miR-31 promoter by the DNA binding factor ETS-1, and it represses miR-31 transcription by  delivering the H3K4me3 demethylase JARID1b/PLU-1/KDM5B. [score:1]
These results also demonstrate that miR-31 is an important antagonist of EMSY’s function in breast cancer. [score:1]
Among these, we find that miR-31, an antimetastatic microRNA involved in breast cancer (Valastyan et al., 2009a), is repressed by EMSY. [score:1]
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These results suggest that MIR31 directly binds to LATS2 mRNA and regulates the LATS2 protein expression via translational inhibition. [score:9]
Figure 2 MIR31 regulates the LATS2 expression by inhibiting translation. [score:8]
We found that LATS2 was downregulated in the MIR31 -overexpressing cells, whereas LATS2 was increased by the MIR31-specific inhibitor compared with that observed in the control cells in a Western blot analysis (Figure  2b). [score:7]
Among several candidates, we focused on LATS2 because it is a known tumor suppressor gene that has been previously reported to be a direct target of MIR31 [24, 34]. [score:6]
It is plausible that MIR31 represses the expression of tumor suppressor genes, such as LATS2, to act as an oncomir and indirectly promotes the transcription of genes related to cell cycle control and tumorigenesis (Figure  6). [score:6]
The LATS2 expression was increased in the control cell tumors compared with that observed in the MIR31 -expressing tumor cells, and the CCND1 levels were increased in the tumors formed from MIR31 -expressing cells (Figure  3f). [score:6]
MIR31 reduces the protein levels of LATS2 by inhibiting translation. [score:5]
Click here for file (a) The expression levels of RAS and XIAP in the mock and MIR31 -overexpressing cells (top). [score:5]
These results suggest that the suppression of the LATS2 expression induced by MIR31 contributes to enhanced tumorigenesis. [score:5]
We found the MIR31 overexpression to be associated with increased CCND1, RAS and XIAP expression levels (Figure  3c, Additional file 8: Figure S8a top) and they also decreased in the anti-MIR31-oligonucleotide -induced cells (Figure  3c, Additional file 8: Figure S8a bottom). [score:5]
The LATS2 expression was increased by the MIR31-specific inhibitor (bottom). [score:5]
A total of 200 nM of miRIDIAN microRNA Hairpin Inhibitor and its negative control (Thermo Scientific Dharmacon, Lafayette, CO, USA) were employed to transiently inhibit MIR31 and transfected 48 hours prior to seeding with Oligofectamine (Invitrogen). [score:5]
In previous studies that reported MIR31 to be an oncomir, MIR31 was found to regulate RAS p21 GTPase Activating Protein 1 (RASA1) [22] and RhoBTB1 [36] in colorectal cancer, LATS2 and PP2A regulatory subunit B alpha isoform (PPP2R2A) [24] in lung cancer and factor-inhibiting hypoxia-inducible factor (FIH) [26] in head and neck carcinomas. [score:5]
To confirm that LATS2 is a target of MIR31 in HEC-50B cells, the protein levels of LATS2 were analyzed in HEC-50B cells overexpressing MIR31. [score:5]
We established HEC-50B cells overexpressing MIR31 by introducing precursor-MIR31 using lentivirus vectors because the MIR31 expression level of HEC-50B was modest among the several adenocarcinoma cell lines analyzed (Additional file 1: Figure S1) and lentivirus vectors can be efficiently transfected into this cell line (HEC-50B mock and MIR31). [score:5]
When we divided the 34 patients into two groups according to the MIR31 expression (MIR31/RNU44 = 15), the MIR31 expression levels were found to be low in the LATS2 -positive (73%) and CCND1 -negative (27%) tumors and high in the LATS2 -negative (25%) and CCND1 -positive (75%) tumors. [score:5]
Because we hypothesized that nuclear YAP1 promotes the transcription of several anti-apoptosis and pro-proliferation genes, we analyzed the expression levels of several proteins, including CCND1, RAS, XIAP, cyclin E1, MYC, KIT, JNK, AKT, FAS, FADD, FASLG and BCL2, in the HEC-50B MIR31 -overexpressing and control cells using immunoblotting (Additional file 7: Figure S7). [score:5]
We next performed a luciferase reporter assay to assess whether MIR31 inhibits the translation of LATS2. [score:4]
MIR31 indirectly promotes the translocation of YAP1 into the nucleus by repressing the tumor suppressor gene LATS2. [score:4]
We compared the MIR31 expression quantified by and the immunohistochemical expression of LATS2 and CCND1 in 34 EC patients who underwent surgery as their initial treatment (Table  1, Lane 1). [score:4]
Additionally, the connection between MIR31 and the p53 mutation could therefore explain the reason why MIR31 either promotes or suppresses different cancers. [score:4]
Furthermore, the expression levels of MIR31 were significantly increased (10.7-fold) in the patients (n = 27) with a high risk of recurrence compared to that observed in the low-risk patients (n = 7), and this higher expression correlated with a poor survival. [score:4]
As MIR31 tends to block the cell apoptosis induced by ultraviolet treatment in HEC-50B cells (data not shown), we speculate that other transcriptional targets of YAP1 associated with apoptosis, such as XIAP, may be coordinately regulated with CCND1. [score:4]
The detection of a normalized luciferase activity revealed that MIR31 significantly suppressed the activity of luciferase combined with wild-type LATS2 3’-UTR in the HEC-50B MIR31 cells, whereas no differences were observed following treatment with the control luciferase and LATS2 3’-UTR possessing a mutation in the putative MIR31 -binding site (Figure  2c). [score:4]
Overexpression of MIR31. [score:3]
The overexpression of MIR31 enhances tumorigenesis in vitro and in vivo. [score:3]
As no significant differences in the LATS2 mRNA levels were observed between the HEC-50B control and MIR31 -overexpressing cells (Additional file 4: Figure S4), MIR31 does not appear to degrade LATS2 mRNA. [score:3]
Precursor-MIR31 was transfected into HEC-50B using the BLOCK-iT™ Lentiviral miR RNAi Expression System (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol, as previously described [45]. [score:3]
Although MIR31 overexpression did not affect in vitro cell proliferation under the standard culture conditions (data not shown), it significantly promoted colony formation under serum starvation (Figure  1b). [score:3]
The MIR31 expression is increased in high-risk human endometrial cancers. [score:3]
Correlations between the MIR31, LATS2 and CCND1 expression in EC. [score:3]
Furthermore, MIR31 has been reported to be an oncomir in various human cancers, including colorectal [22], esophageal [23], lung [24], oral [25] and head and neck [26] cancer, and a tumor suppressor gene in breast [27] and gastric [28] cancers and malignant mesothelioma [29]. [score:3]
The cells successfully transfected with control or precursor MIR31 vectors expressed Green Fluorescent Proteins (Figure  3a top), and YAP1 was stained with Cy5 (Figure  3a bottom). [score:3]
We confirmed the MIR31 expression in three EC cell-lines, HEC-50B, HEC-1A and HEC-108, using and found that the MIR31 levels were lowest in the HEC-108 cells, followed by HEC-1A and HEC-50B cells (Additional file 1: Figure S1, Lanes 1, 2 and 3). [score:3]
Therefore, when LATS2 is suppressed by MIR31, it is expected that the translocation of YAP1 into the nucleus would be promoted. [score:3]
We found that the expression of MIR31 was significantly increased in the high-risk patients (Figure  4d). [score:3]
The presence of a mature-MIR31 expression was confirmed using (Figure  1a). [score:3]
MIR31 is an oncomir in several human cancers and a tumor suppressor gene in others. [score:3]
Additionally, an MIR31-specific inhibitor significantly restrained colony formation (Additional file 3: Figure S3). [score:3]
Click here for file Correlation between the MIR31 expression and risk of postoperative recurrence in the patients with grade 2 tumors. [score:3]
The overexpression of MIR31 significantly promoted anchorage-independent growth in vitro and significantly increased the tumor forming potential in vivo. [score:3]
The results of the analysis of the expression levels of MIR31 are shown in the bar graph. [score:3]
Figure 1 The overexpression of MIR31 enhanced tumorigenesis in vitro and in vivo. [score:3]
The colony number was increased in the same order as the MIR31 expression under two different serum concentrations (Additional file 2: Figure S2). [score:3]
analysis of the MIR31 expression in five adenocarcinoma cell lines of the female genital tract. [score:3]
Click here for file analysis of the MIR31 expression in five adenocarcinoma cell lines of the female genital tract. [score:3]
We also found a correlation between the MIR31, LATS2 and CCND1 expression in vivo (mouse 7 in Figure  1c). [score:3]
The RAS and XIAP levels were decreased by the MIR31-specific inhibitor (bottom). [score:3]
The MIR31 expression was lowest in the LATS2 -positive and CCND1 -negative groups and highest in the LATS2 -negative and CCND1 -positive groups (Figure  4c). [score:3]
In order to elucidate the mechanisms by which MIR31 promotes tumorigenesis, in silico prediction mo dels were employed to identify the target mRNAs of MIR31 [10]. [score:3]
Mock and MIR31 cells were transfected with non -targeting siRNA. [score:3]
MIR31 significantly suppressed the luciferase activity of mRNA combined with the LATS2 3’-UTR and consequently promoted the translocation of YAP1, a key molecule in the Hippo pathway, into the nucleus. [score:3]
In this study, we divided 34 EC patients into two groups according to the criteria generally used to determine whether postoperative adjuvant therapy is required and found a strong correlation between the MIR31 expression and these clinical risk factors. [score:3]
The primers used for the expression analysis were as follows: pre- MIR31 - forward, 5'-GGAGAGGAGGCAAGATGCTG-3'; pre- MIR31 - reverse, '-GGAAAGATGGCAATATGTTG-3': GAPDH - forward, 5'-CTCATGACCACAGTCCATGC-3': GAPDH - reverse, 5'-TTACTCCTTGGAGGCCATGT-3': LATS2 - forward, 5'-TAGAGCAGAGGGCGCGGAAG -3': LATS2 - reverse, 5'- CCAACACTCCACCAGTCACAGA-3'. [score:2]
A significant increase in tumor weight was observed in the HEC-50B cells with MIR31 overexpression compared with that noted in the controls in the nude mice subcutaneous tumor mo del (Figure  1c). [score:2]
Because these results strongly suggest that nuclear YAP1, which is increased by the MIR31 expression, promotes the transcription of these targets, we performed luciferase reporter assays in order to investigate the influence of MIR31 on the transcription of CCND1, RAS and XIAP. [score:2]
In this study, we aimed to investigate whether MIR31 is an oncomir in human EC and identify the direct target associated with the malignant phenotype of EC. [score:2]
As expected, we found that the nuclear translocation of YAP1 frequently occurred in the MIR31 -overexpressing cells compared with that observed in the control cells (Figure  3b). [score:2]
In order to identify the target molecule of MIR31, a luciferase reporter assay was performed, and the corresponding downstream signaling pathway was examined using immunohistochemistry of human endometrial cancer tissues. [score:2]
MIR31 promotes the transcription of cyclin D1 (CCND1) via dysregulation of the Hippo signaling pathway. [score:2]
The detection of a normalized luciferase activity revealed that the MIR31 expression significantly increased the activity of luciferase driven by the CCND1, RAS and XIAP promoters compared with that observed in the control cells (Figure  3d Line 1–2, Additional file 8: Figure S8b). [score:2]
Our findings suggested the existence of a possible connection between MIR31 and p53 mutation in EC which thus induces the aggressiveness of EC. [score:2]
MIR31 was detected using quantitative real-time PCR (qRT-PCR). [score:1]
MIR31 is a potential new molecular marker for predicting the risk of recurrence and prognosis of endometrial cancer. [score:1]
We also investigated the MIR31 expression in 34 patients according to the postoperative risk of recurrence. [score:1]
Figure 5 Prognosis of patients classified into the high and low MIR31 groups. [score:1]
MIR31 is not involved in YAP phosphorylation. [score:1]
We speculate that MIR31 has a specific function in each type of malignancy, and several mechanisms, including methylation -dependent silencing [35] and local deletion [29], may explain its different roles in different tumor types. [score:1]
We investigated the growth potentials of MIR31 -overexpressing HEC-50B cells in vitro and in vivo. [score:1]
MIR31 is correlated with enhanced colony formation of EC cell lines. [score:1]
These results suggest that MIR31 is potentially a new molecular marker for distinguishing the risk of recurrence combined with histological findings. [score:1]
One potential binding site for MIR31 was found in the 3’-UTR region of LATS2 mRNA (Figure  2a). [score:1]
Figure 3 MIR31 promotes the translocation of YAP1 into the nucleus and promotes the transcription of CCND1. [score:1]
MIR31 functions as an oncogene in endometrial cancer by repressing the Hippo pathway. [score:1]
Six-week-old BALB/c nude mice (Clea, Tokyo, Japan) were injected subcutaneously into their flanks with 2 x 10 [7] HEC-50B mock or HEC-50B MIR31 cells bilaterally in 200 μl of normal culture medium. [score:1]
These results suggest that MIR31 is related to the aggressiveness of EC. [score:1]
We aimed to investigate whether MIR31 is an oncogene in human endometrial cancer and identify the target molecules associated with the malignant phenotype. [score:1]
Click here for file MIR31 is not involved in YAP phosphorylation. [score:1]
As expected, the progression-free survival was significantly worse among the patients with high MIR31 tumors (> = 0.8) than among those with low MIR31 tumors (<0.8) (Figure  5). [score:1]
These results suggest that the translocation of YAP1 into the nucleus is the most important effect of MIR31. [score:1]
In this study, we demonstrated that MIR31 functions as an oncomir in EC. [score:1]
However, little is known about the biological functions of MIR31 in EC. [score:1]
In conclusion, we herein demonstrated that MIR31 promotes EC tumorigenesis. [score:1]
The MIR31 -mediated tumorigenic effects were confirmed in an in vivo mo del. [score:1]
These findings demonstrate that MIR31 induces a more aggressive phenotype of EC. [score:1]
*The translocation of YAP1 into the nucleus was not observed in the cells unsuccessfully transfected with pre-MIR31. [score:1]
However, little is known about the MIR31 status in patients with EC. [score:1]
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In addition to suppressing GNA13 expression, we observed that miR-31 suppresses invasion of MDA-MB-231 cells, and that this could be partially rescued upon ectopic expression of miR-31-resistant GNA13 in the cells. [score:9]
Further, expression of miR-31 significantly inhibited MDA-MB-231 cell invasion, and this effect was partly rescued by ectopic expression of GNA13 in these cells. [score:7]
However, in our system miR-31 neither targeted the RhoA-3′-UTR, nor did RhoA protein expression correlate to miR-31 expression in these breast cancer cells (Additional file 1: Figure S1 and Figure S2). [score:7]
RhoA protein expression in breast cancer cells: RhoA protein expression showed no correlation to basal miR-31 expression in breast cancer cells (see Figure  3C for miR-31). [score:7]
A previous report showed that enforced expression of miR-31 inhibits invasion and metastasis of breast cancer cells, and also that this miRNA is suppressed in metastatic breast cancers [36]. [score:7]
Additionally our data shows that miR-31 regulates breast cancer cell invasion partially via targeting GNA13 expression in breast cancer cells. [score:6]
Hence, in addition to demonstrating that miR-31 directly binds to the GNA13-3′-UTR and inhibits GNA13 expression, these data suggest that miR-31 -induced degradation of the mRNA may play a role in this process. [score:6]
However, analysis of the basal expression of other miRNAs that are predicted to bind to GNA13-3′-UTR revealed that miR-31 showed an inverse correlation to GNA13 expression in these cells, and further studies showed that miR-31 was indeed was involved in control of GNA13 levels in the breast cancer cells. [score:5]
Figure 5 miR-31 inhibits GNA13 expression and cancer cell invasion. [score:5]
Importantly, miR-31 -induced suppression of invasion was significantly rescued by enforced expression of GNA13 in premiR-31 treated cells (Figure  5D, E). [score:5]
GNA13 mRNA expression shows an inverse correlation to pri-miR-31 expression in breast cancer tissues. [score:5]
Altering miR-31 expression or activity impacts GNA13 mRNA and protein expression in MDA-MB-231 and MCF-10a cells. [score:5]
MDA-MB-231 and MDA-MB-157 cells expressed high GNA13 protein and had no or little detectable miR-31 expression (Figure  3A, B). [score:5]
Expression analysis of miRNAs predicted to bind the 3′-UTR of GNA13 revealed that miR-31 exhibited an inverse correlation to GNA13 protein expression in breast cancer cells. [score:5]
To determine whether miR-31 was the inhibitory element expressed in MCF-10a cells, antimiR-31 was co -transfected with the GNA13-3′-UTR into the cells. [score:5]
This partial rescue of miR-31 -induced suppression of invasion reinforces the notion that miR-31 has multiple targets that contribute to invasion of these cells. [score:5]
A screen for microRNAs that are predicted to target GNA13 in breast cancer cells revealed that miR-31 shows an inverse correlation to GNA13 protein expression in these cells. [score:5]
These data collectively indicate that miR-31 inhibits invasion of MDA-MB-231 cells and that this is at least in part mediated through an impact on GNA13 expression. [score:5]
MiR-31 is an example of a tumor suppressor miR, and is a pleotropically acting miRNA that targets multiple oncogenes such as integrin-alpha5, radixin, and EZH2 [32, 33]. [score:5]
In addition, inhibition of miR-31 expression using antimiR-31 in MCF-10a cells resulted in a significant increase in GNA13 mRNA and protein levels (Figure  5A, B). [score:5]
Inhibition of miR-31 activity by such enforced expression of antimiR-31 largely rescued the GNA13-3′-UTR and the miR-31 sensor (a reporter carrying miR-31 binding site [35]) activity in these cells (Figure  4C). [score:5]
Loss of miR-31 expression and increased GNA13 expression could be used as biomarkers of breast cancer progression. [score:5]
Most importantly, a correlation analysis of miR-31 expression to GNA13 expression showed a significant inverse correlation, as shown in Figure  6B. [score:5]
Surprisingly, unlike prostate cancer cells, GNA13 expression in breast cancer cells is mainly regulated through miR-31 and not through miR-182 and miR-200a. [score:4]
This data provide compelling evidence that miR-31 directly binds to the GNA13-3′-UTR and inhibits its activity. [score:4]
To determine whether, miR-31 is actually involved in the regulation of expression of GNA13 in these cells, we transfected MDA-MB-231 cells with premiR-31, and MCF-10a cells with antimiR-31, respectively. [score:4]
These data provide strong evidence that GNA13 expression in breast cancer cells is regulated by post-transcriptional mechanisms involving miR-31. [score:4]
Our data demonstrate that GNA13 is an important mediator of cancer cell invasion, and that its expression is regulated by microRNA-31 in breast cancer cells. [score:4]
MiR-31 activity reduces GNA13 mRNA and protein expression and inhibits breast cancer cell invasion. [score:4]
The data presented above provide compelling evidence that miR-31 directly binds to GNA13 mRNA, and that this binding impacts both GNA13 expression and breast cancer cell invasion. [score:4]
In our screen for the expression of miR-31 and GNA13 mRNA in panel of 41 breast tumor tissues, mRNA levels for miR-31 showed an inverse correlation to those of GNA13. [score:3]
Relative miR-31 expression after transfection with premiR-31 and antimiR-31 respectively is shown in Figure  5C. [score:3]
These data reinforce a previous finding that loss of miR-31 might be an important determinant in breast cancer metastasis [33, 36], and further suggest that gain of GNA13 expression might be one of the important contributors to the phenotype. [score:3]
The tissues were divided into two groups based on their Pri-miR-31 expression as miR-31 high (n = 16) and miR-31 low (n = 22), respectively. [score:3]
In addition, altering the endogenous miR-31 levels using premiR-31 or antimiR-31 significantly altered GNA13 mRNA and protein expression in MDA-MB-231 and MCF-10a cells respectively. [score:3]
These data provide evidence that microRNA-31 might indeed be one of the determinants of GNA13 expression in breast cancers. [score:3]
Ectopic expression of miR-31 in MDA-MB-231 cells significantly reduced GNA13 mRNA and protein levels, as well as GNA13-3′-UTR-reporter activity. [score:3]
Interestingly, however, miR-31 showed a clear inverse correlation to GNA13 protein expression in the breast cancer cells (Figure  3A, B). [score:3]
Importantly a screen for miR-31 and GNA13 expression showed a significant inverse correlation of these two transcripts in breast cancer tissues. [score:3]
Previous reports have shown that miR-31 expression is lost during cancer progression in breast and other cancer types, either by genetic or epigenetic mechanisms [33, 48]. [score:3]
To obtain evidence as to whether miR-31 binds to the 3′-UTR of GNA13 as suggested by the data presented above, we cloned the full-length UTR into miR-Sens-reporter where it controls the expression of the Renilla luciferase gene in the vector (see Figure  2B in Rasheed etal [28] for schematic of GNA13-3′-UTR). [score:3]
Taken together, our data indicates that loss of miR-31 in breast cancers leads to increased GNA13 expression and cancer cell invasion. [score:3]
All values were calibrated to basal miR-31 expression in MDA-MB-231 cells. [score:3]
This will enable efficient binding of miR-31 to this sequence and suppress the Renilla luciferase activity. [score:3]
Specifically in this regard, miR-31 has been implicated in targeting multiple genes linked to cell invasion, e. g., integrin-alpha5, radixin, RhoA, EZH2, PRKCE and LATS2 [32, 33, 35, 47]. [score:3]
All of the data presented to date are consistent with the notion that the GNA13-3′-UTR is a target of miR-31 activity in breast cancer cells. [score:3]
Consistent with this finding, transfection of premiR-31 into MDA-MB-231 cells suppressed the activities of both the GNA13-3′-UTR and the miR-31 sensor (Figure  4D). [score:3]
Since GNA13 is known to promote invasion mainly through activating RhoA [28], we also studied the impact of miR-31 on RhoA expression. [score:3]
GNA13-3′-UTR activity assays showed that miR-31 directly binds to the 3′-UTR and suppressed its activity. [score:3]
These data implicate miR-31 in control of GNA13 expression in breast cancer cells. [score:3]
Post-transcriptional regulation Gα13 G12 proteins Invasion miR-31 Breast cancer In spite of tremendous progress in cancer therapy, breast cancer remains one of the major causes of female mortality due to cancer. [score:2]
Comparing the basal GNA13 mRNA expression in these two groups showed that miR-31 high tissues had significantly less GNA13 mRNA as compared with miR-31 low tumors (Figure  6A). [score:2]
Briefly, miR-31 sensor is created by direct ligation of annealed oligos at Xho1-Not1 sites in miR-Sens vector downstream to Renilla luciferase reporter. [score:2]
Further, loss of miR-31 and gain of GNA13 may be viable biomarkers for assessment of breast and other cancers. [score:1]
Values reported are relative to miR-31 levels in MDA-MB-231 cells treated with premiR-control. [score:1]
, miR-Sens-MUT-31 and miR-Sense-miR-31-Sensor was produced using the pCL-Ampho® amphotropic virus packaging plasmid in HEK293T cells as described previously [35]. [score:1]
Examination of 48 human breast cancer tissues revealed that GNA13 mRNA levels were inversely correlated to miR-31 levels. [score:1]
This sensor carries a single miR-31 binding site as the 3′-UTR of Renilla Luciferase reporter. [score:1]
Conversely, blocking miR-31 activity in MCF-10a cells induced GNA13 mRNA, protein and 3′-UTR reporter activity. [score:1]
miR-31-Sensor’s efficiency and specificity are already tested and described in Beillard et al. [35]. [score:1]
Mir-31 binds directly to the 3′UTR of GNA13. [score:1]
Relationship of microRNA-31 and GNA13 in breast cancer cells. [score:1]
Relationship of miR-31 and GNA13 in breast cancer tissues. [score:1]
It is important to note that miR-31 and miR-182 had overlapping binding sites, suggesting that either of these miRNAs could bind to the site based on the availability of the miRNA. [score:1]
Construction of GNA13-3′-UTR and miR-31-sensor-luciferase plasmids. [score:1]
Further, when the predicted binding sequence for miR-31 in the GNA13-3′-UTR was mutated; neither antimiR-31 nor premiR-31 had any effect on the reporter activity (Figure  4C, D). [score:1]
Oligos used to clone miR-31-Sensor in miR-Sens vector as described in Beillard et al. Figure S1. [score:1]
Primers used in the cloning of GNA13-3′-UTR and for site-directed mutagenesis of miR-31 binding sites within the UTR. [score:1]
Any change in miR-31 levels is efficiently reflected as change in reporter activity. [score:1]
Figure 3 Relationship of miR-31 and GNA13 in breast cancer cells. [score:1]
The oligos carry a sequence complementary to mature miR-31 sequence. [score:1]
The miRNAs that were predicted to bind the GNA13-3′ -UTR are miR-30 family, miR-27a/b, miR-128, miR-31, miR-182, miR-29a/b/c and miR-141/200a. [score:1]
MicroRNA mimics (PremiRs) for miR-31, the PremiR-control, and antimiR-31 and the antimiR-control were purchased from Qiagen. [score:1]
The oligo sequences used to create miR-31 Sensor are provided in the 1: Table S1. [score:1]
Most importantly, multiple studies have shown that miR-31 is lost during cancer progression and promotes metastasis of breast and other cancers [33, 34]. [score:1]
To begin to address this question, we performed an analysis of mRNA levels of Pri-miR-31 (primary transcript of miR-31) and GNA13 in 7 normal breast and 41 breast tumor tissues. [score:1]
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Other miRNAs from this paper: hsa-mir-216a, hsa-mir-217, hsa-mir-489
Our study showed that miR-31 induced EMT via regulating BAP1 expression in cervical cancer cells and marked the expression of E-cadherin downregulated and N-cadherin and vimentin upregulated. [score:12]
Moreover, downregulation of N-cadherin and vimentin and upregulation of E-cadherin by miR-31 inhibitor were also rescued by cotransfected inhibition of miR-31 and BAP1 (Figure 6(c)). [score:11]
We also detect the mRNA and protein expression level of BAP1, tumors with BAP1 plasmid transfected group exhibited higher BAP1 mRNA and protein expression compared with the control group (Figures 4(c) and 4(d)), suggesting that BAP1 plasmid overexpression could rescue the expression of BAP1 suppressed by miR-31. [score:10]
BAP1 was a direct target of miR-31; downregulation of BAP1 by miR-31 -induced EMT in cervical cancer. [score:7]
BAP1 may function as a tumor suppressor in cervical cancer; it is very significant to inhibit miR-31 to induce BAP1 expression. [score:7]
To gain further insight into the regulatory effect of miR-31-BAP1 on EMT in cervical cancer, miR-31 inhibitor and BAP1 siRNA were cotransfected into HeLa and C33A cells to simultaneously silence miR-31 and BAP1 expression. [score:6]
In this study, we showed that BAP1 was a direct target of miR-31 and an inverse correlation between miR-31 and BAP1 expression in cervical cancer cells and tissues. [score:6]
Upregulation of miR-31 and Knockdown of BAP1 Promoted Cell Migration In Vitro. [score:5]
In summary, we demonstrated that miR-31 can induce EMT by downregulating BAP1 in cervical cancer, and miR-31 -induced acceleration of cellar migration and invasion was partially by its regulation of BAP1 in vitro and in vivo. [score:5]
In agreement with the previous studies, miR-31 expression was detected significantly upregulated in cervical cancer tissues compared with nontumor tissues (Figures 1(a) and 1(b)). [score:5]
Inhibition of miR-31 expression enhanced the level of BAP1 in the two cell lines (Figures 2(c) and 2(d)). [score:5]
Xenograft tumor experiment observed the effect of miR-31 on EMT, which indicated that overexpression BAP1 plasmid inhibits the tumor growth. [score:5]
As previously reported, HeLa cell was miR-31 overexpression cell line and therefore we transfected HeLa cells with a BAP1 overexpression plasmid. [score:5]
Yu et al. found that BAP1 was a target of miR-31 and inhibited lung cancer progression [19]. [score:5]
The human cervical cancer cells were seeded at 6-well plate and transfected with miR-31, miR-31 negative control (miR-NC), miR-31 inhibitor (anti-miR-31), or miR-31 inhibitor negative control (anti-miR-NC) using Lipofectamine 2000 (Invitrogen) on the following day when the cells were approximately 60–70% confluent, following the manufacturer's instructions. [score:5]
As expected, inhibition of miR-31 suppressed cell migration ability (Figures 3(a) and 3(b)). [score:5]
To date, miR-31 has been reported as upregulated in cervical cancer, which implies that miR-31 may serve as an onco-miRNA participating in the development of cervical cancer [12]. [score:5]
In vivo and in vitro results revealed that the oncogenic miR-31 inhibitor inhibits the tumor growth and cell metastasis. [score:5]
These results indicate that miR-31 binds to BAP1 3′-UTR directly and negatively regulates BAP1 expression in cervical cancer cells. [score:5]
In this study, we first identified that miR-31 expression is inversely correlated to the expression of BAP1. [score:5]
The results above suggest that BAP1 overexpression reverses the effect of miR-31 on tumor growth and miR-31 could promote tumor growth by silencing BAP1 expression in cervical cancer. [score:5]
Confirming That BAP1 Is a Direct Target of miR-31. [score:4]
Recent reports suggest that aberrant expression of miR-31 plays crucial role in human cancer development, and miR-31 served as an oncogene in lung and colorectal cancer [20, 21]. [score:4]
This suggested that miR-31 may serve as a direct therapeutic target for cervical cancer patients. [score:4]
In cervical cancer cases, upregulation of miR-31 was found to be correlated with shorter survival time and poorer prognosis. [score:4]
HeLa cells were cultured in 24-well plate and cotransfected with miR-31 and BAP1 3′-UTR wild type or mutation of the putative miR-31 target region using Lipofectamine 2000 (Invitrogen). [score:4]
Our clinicopathological data also showed that upregulation of miR-31 was significantly associated with advanced clinical stage, tumor size, lymph node metastasis, and deep stromal invasion. [score:4]
3.1. miR-31 Is Upregulated and Correlated with Poor Survival in Cervical Cancer. [score:4]
These results indicated that miR-31 directly targeted BAP1 and induced EMT-like changes in cervical cancer. [score:4]
Upregulation of miR-31 and Knockdown of BAP1 Promoted Cell Migration In VitroTo explore the role of miR-31-BAP1 in cervical cancer metastasis, Transwell migration and wound healing assay were performed in HeLa, C33A, and HaCaT cells. [score:4]
By transient cotransfection of miR-31 with BAP1 3′-UTR wild type or mutation of the putative miR-31 target region into HeLa cells, the results showed that miR-31 significantly decreased activity of the luciferase reporters with WT 3′-UTR, but the activity of MUT 3′-UTR vector remained unaffected (Figure 2(b)). [score:4]
Next, we confirmed the inverse relationship between miR-31 and BAP1 expression in HeLa and HaCaT cells. [score:3]
Among the candidate genes, BAP1 was predicated as a target of miR-31 by all four algorithms and was selected for experimental verification. [score:3]
Prediction of BAP1 as a Target of miR-31. [score:3]
We use quantitative real-time PCR to confirm the altered expression level of miR-31 in cervical cancer tissues and adjacent nontumor tissues. [score:3]
We found that dual knockdown of BAP1 and miR-31 significantly reversed cell migration ability of HeLa and C33A cells induced by single-knockdown of miR-31 (Figures 6(a) and 6(b)). [score:3]
In general, miR-31 interference with BAP1 recovery expression may be a potential therapeutic strategy for metastatic cervical cancer patients. [score:3]
Therefore, our study clarified previously unidentified prometastatic roles of miR-31 in cervical cancer and miR-31-BAP1 pathway might be a new potential target for therapy in cervical metastasis. [score:3]
In order to investigate the molecular mechanism by which miR-31 executed its function, four bioinformatics algorithms (TargetScan, miRanda, Microcosm, and PicTar) were used in combination to identify the more accurate potential targets of miR-31. [score:3]
To detect BAP1 as the direct binding target of miR-31, a luciferase reporter assay was performed. [score:3]
The results show that upregulation of miR-31 increased tumor cell migratory ability compared with the negative control cells. [score:3]
We further examined whether BAP1 was a direct target of miR-31 in cervical cancer cells using dual-luciferase reporter assay. [score:3]
Furthermore, the clinicopathological parameters showed that increased expression of miR-31 was correlated with node metastasis, deep stromal invasion, vascular involvement, and FIGUREO stage and tumor size, but there was no significant correlation between miR-31 level and age and tumor size (Table 1). [score:3]
Kaplan-Meier analysis revealed that patients with high miR-31 had less overall survival times than those with low miR-31 expression (Figure 1(c)). [score:3]
Silencing of BAP1 Reverses the Antitumor Effects of miR-31 Inhibitor. [score:3]
Thus, miR-31 functions as an oncogene or tumor suppressor dependent on the cellular types and contexts. [score:3]
Conversely, miR-31 may also act as a tumor suppressor in breast cancer [23], bladder cancer [10], and prostate cancer [11]. [score:3]
Among these miRNAs, miR-31 may function as an oncogene or a tumor suppressor depending on the cellular contexts [8– 11]. [score:3]
miR-31: MicroRNA-31 BAP1: BRCA1 -associated protein-1 EMT: Epithelial-mesenchymal transition HPVs: High-risk human papillomaviruses 3′UTR: 3′ untranslated region WT: Wild type MUT: Mutant type PVDF: Polyvinylidene fluoride IHC: Immunohistochemical qRT-PCR: Quantitative reverse transcriptional PCR. [score:3]
To gain further insight into whether miR-31 was an EMT- regulatory miRNA in cervical cancer, Western blot analysis was performed to examine the EMT-related molecules in cervical cancer cells. [score:2]
We also validated that BAP1 could promote cervical carcinogenesis and EMT progression was regulated by miR-31. [score:2]
It needs a lot of work to further investigate the mechanism of miR-31 upregulated in cervical cancer. [score:2]
Taken together, these data indicate that BAP1 is a key effector of invasion and migration in cervical cancer, which was regulated by miR-31. [score:2]
3.6. miR-31-BAP1 Signaling Promotes Cell Migration by Stimulating EMT. [score:1]
The 3′-UTR of BAP1 contained a high conserved binding site for miR-31 seed region as shown in Figure 2(a). [score:1]
In the present study, our findings indicate that miR-31 functions as an oncogenic miRNA in the progress of cervical carcinogenesis. [score:1]
However, there has very little research on the role of BAP1 in cervical cancer and the relationship between miR-31-BAP1 and EMT has not yet been reported. [score:1]
The Influence of miR-31 and BAP1 on the Growth of Cervical Cancer Cells In Vivo. [score:1]
Whether miR-31 can induce EMT or other malignant transformation in cervical cancer remains unknown. [score:1]
Taken together, these results indicate that miR-31 was involved in the progression and invasion/metastasis of cervical cancer and was correlated with worse prognosis. [score:1]
According to the previous published papers, the role and mechanism of miR-31 in cervical cancer are mainly confined to tumor growth. [score:1]
miR-31 has also been found to behave as an oncogenic miRNA in other human cancers, including lung cancer [20], colon cancer [21], head and neck cancer [22], and hepatocellular carcinoma [9]. [score:1]
Taken together, these data indicate that miR-31-BAP1 plays important role in the proliferation of cervical cancer cells. [score:1]
Due to the complex effect of miR-31, our study aimed to identify its potential biological function in cervical cancer. [score:1]
5 × 10 [4] cells/well transfected with miR-31 or miR-NC were seeded into the upper chamber of 24-well Transwell plates containing 1% FBS medium. [score:1]
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[+] score: 231
Other miRNAs from this paper: hsa-let-7b, hsa-mir-26a-1, hsa-mir-26a-2, hsa-mir-506
A recent study has reported that EZH2 suppresses miR-31 expression by inducing H3K27me3 on the miR-31 promoter and that the inhibition of EZH2 increased miR-31 expression in prostate cancer [2]. [score:9]
In addition, we demonstrated that the knockdown of EZH2 by siRNAs increased miR-31 expression in colon cancer cell lines, suggesting that negative EZH2 expression causes miR-31 upregulation in the progression of SSA/Ps. [score:9]
Therefore, to determine if EZH2 is involved in the regulation of miR-31 expression through histone H3K27me3, we examined H3K27me3 levels around the transcription start site on the miR-31 promoter by performing ChIP assay and found that EZH2 -mediated histone methylation downregulates miR-31 expression in colon cancer. [score:8]
The present multivariate analysis showed that high EZH2 expression was inversely associated with miR-31 expression in colorectal cancer, whereas functional analysis showed that EZH2 knockdown increased miR-31 expression in colon cancer cell lines. [score:8]
Male) 1.78 (1.08-2.98) 0.025 A multivariate logistic regression analysis assessing the relationships with EZH2 expression status initially included gender, age, tumor size, year of diagnosis, tumor location, tumor differentiation, disease stage, CIMP status, MSI status, mutations of BRAF, KRAS and PIK3CA, and microRNA-31 expression, considering potential confounding and causal relationships. [score:8]
Taken together with increased expression of miR-31 after EZH2 knockdown (Figure 4C), these results suggest the expression of miR-31 is suppressed by EZH2 through H3K27me3 in R KO cells. [score:8]
In conclusion, we identified an inverse association between the expressions of EZH2 and miR-31 in colorectal cancer and that the upregulation of EZH2 expression may be a rare event in SSA/Ps. [score:8]
EZH2 reportedly downregulates miR-31 expression in human cancers such as prostate cancer [2] and adult T-cell leukemia [16]. [score:6]
Thus, accumulating evidence indicates that EZH2 may downregulate miR-31 expression in human cancers; however, no study has reported the relationship between EZH2 and miR-31 in colorectal cancer. [score:6]
A multivariate logistic regression analysis assessing the relationships with EZH2 expression status initially included gender, age, tumor size, year of diagnosis, tumor location, tumor differentiation, disease stage, MSI, CIMP, mutations of BRAF, KRAS and PIK3CA, and miR-31, considering potential confounding and causal relationships. [score:6]
To examine whether EZH2 suppressed miR-31 expression, we knocked down EZH2 mRNA by siRNAs in colon cancer cell lines and measured the resulting miR-31 expression. [score:6]
However, EZH2 expression was inversely associated with miR-31 expression (P < 0.0001) (Table 1 and Figure 2). [score:5]
Recent evidence has shown that microRNAs can act as both oncogenes and tumor suppressors, depending on the genes they regulate [6, 17]; for example, microRNA-31 (miR-31) is reportedly deregulated in human cancers [1, 2, 6, 16, 27, 28, 30, 35], and provides oncogenic potential in colorectal cancer [27- 29, 31, 36]. [score:5]
Furthermore, we performed functional analyses to identify whether EZH2 suppressed miR-31 expression in colorectal cancers. [score:5]
Figure 2EZH2 expression levels were inversely associated with microRNA-31 expressions in colorectal cancers (P < 0.0001). [score:5]
Low expression group (score 0-2)] Adjusted odds ratio (95% CI) P High microRNA-31 expression (vs. [score:5]
Furthermore, EZH2 -mediated histone methylation has been shown to suppress miR-31 expression in adult T-cell leukemia [16]. [score:5]
The association between EZH2 expression and microRNA-31 expression in 301 colorectal cancers. [score:5]
Therefore, because of the relationship with miR-31, EZH2 may represent a new prognostic biomarker for molecular targeted therapies and can provide a promising therapeutic target in patients with colorectal cancer. [score:5]
EZH2 expression levels were inversely associated with microRNA-31 expressions in colorectal cancers (P < 0.0001). [score:5]
In a database comprising 301 patients with colorectal cancer, high EZH2 expression was inversely associated with miR-31 expression, independent of clinicopathological and molecular features. [score:5]
Conversely, EZH2 has been reported to suppress miR-31 expression by inducing H3K27me3 on the miR-31 promoter in prostate cancer. [score:5]
These results are reasonable because we recently reported that high miR-31 expression, which is inversely correlated with EZH2 expression, is an unfavorable prognostic factor in patients with colorectal cancer [28]. [score:5]
Hence, we suggest that EZH2 suppresses miR-31 expression in colorectal cancer and may correlate with differentiation and evolution of the serrated pathway. [score:5]
Using microRNA array analysis, we recently identified that miR-31 expression was significantly upregulated in BRAF-mutated colorectal cancer compared with wild-type colorectal cancer [28]. [score:5]
Nevertheless, our multivariate regression analysis was adjusted for potential confounders, including clinical and molecular features, and we were able to show that high EZH2 expression was inversely associated with miR-31 expression and associated with better prognosis. [score:5]
In the final mo del, high EZH2 expression (score 3) was inversely associated with high miR-31 expression (Q4) [odds ratio (OR): 0.22; 95% CI: 0.11–0.42; P < 0.0001] (Table 2). [score:5]
To the best of our knowledge, this is the first report describing the downregulation of miR-31 by EZH2 in colorectal cancer. [score:4]
EZH2 knockdown caused microRNA-31 (miR-31) overexpression on quantitative RT-PCR. [score:4]
However, no study has reported the potential role of EZH2 in the regulation of miR-31 expression in colorectal cancer. [score:4]
In functional analysis, we showed that the siRNA knockdown of EZH2 increased miR-31 expression in colon cancer cell lines. [score:4]
Knockdown of EZH2 increases miR-31 expression in colon cancer cell lines. [score:4]
C. There was a considerable increase in miR-31 expression in R KO cells transfected with EZH2 siRNAs (siEZH2_7644 and siEZH2_7882). [score:3]
We conducted this study to clarify the association of EZH2 expression with miR-31 and epigenetic alterations using a database comprising more than 500 colorectal tumors. [score:3]
The distributions of miR-31 expression in the 301 colorectal cancers were as follows: mean 55.4; median 10.7; standard deviation (SD) 211; range 0.11–2108; interquartile range 3.9–32.2 (Supplementary Figure 2). [score:3]
We also reported that high miR-31 expression may correlate with evolution of the serrated pathway [31, 37]. [score:3]
Moreover, we found that there was a considerable increase in miR-31 expression in R KO cells transfected with EZH2 siRNAs (Figure 4C). [score:3]
We recently reported that high miR-31 expression was more pronounced in SSA/Ps with cytological dysplasia than in other SSA/Ps, but we did not find a significant difference between TSAs with high-grade dysplasia and other TSAs [31]. [score:3]
Moreover, associations were identified between miR-31 expression and poor prognosis in colorectal cancer [28]. [score:3]
absent), and miR-31 (high expression vs. [score:3]
Recently, we also reported that high miR-31 expression was associated with shorter progression-free survival in patients with colorectal cancer treated using anti-epidermal growth factor receptor (EGFR) therapy [27]. [score:3]
We summarized the association of EZH2 expression with miR-31 and cancer-specific survival in Supplementary Figure 6. Our current study had some important limitations, particularly because of its cross-sectional nature and unknown bias (i. e. selection bias). [score:3]
Cases with miR-31 expression were divided into quartiles Q1 (<3.9), Q2 (3.9–10.6), Q3 (10.7–32.1), and Q4 (≥32.2) for further analysis. [score:3]
With regard to melanoma, genetic and epigenetic loss of miR-31 produced a feed-forward EZH2 expression [6]. [score:3]
MicroRNA-31 (miR-31)-5p expression was analyzed by qRT-PCR using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and TaqMan microRNA Assays (Applied Biosystems) as described previously [28]. [score:2]
To clarify the functional link between EZH2 and miR-31 expression, we examined H3K27me3 levels around the promoter region of miR-31 by performing chromatin immunoprecipitation (ChIP) assay. [score:2]
We observed that H3K27me3 was steadily enriched at the promoter region of miR-31 in R KO cells, and the H3K27me3 levels were decreased after knockdown of EZH2 (Supplementary Figure 5). [score:2]
These data imply that miR-31 correlate with the progression of SSA/Ps. [score:1]
ChIP chromatin immunoprecipitation CI confidence interval CIMP CpG island methylator phenotype EGFR epidermal growth factor receptor EZH2 enhancer of zeste homolog 2 FFPE formalin-fixed paraffin-embedded HP hyperplastic polyp HR hazard ratio miR-31 microRNA-31 MSI microsatellite instability MSS microsatellite stable PRC2 polycomb repressive complex 2 qRT-PCR quantitative reverse transcription-PCR SD standard deviation siRNA small interfering RNA SSA/P sessile serrated adenoma/polyp TSA traditional serrated adenoma H3K27me3 histone H3 lysine 27 trimethylation WHO World Health Organization. [score:1]
CIMP, CpG island methylator phenotype; MSI, microsatellite instability; MSS, microsatellite stable; miR-31, microRNA-31; SD, standard deviation. [score:1]
RNA extraction and qRT-PCR of microRNA-31. [score:1]
To calculate the relative expression of miR-31 in each colorectal cancer, 2 [−ΔCT] of cancer tissue was divided by 2 [−ΔCT] of normal tissue, as described previously [28]. [score:1]
miR-31 expression was calculated using the equation 2 [−ΔCT], where ΔC [T] = (C [T] miR-31 − C [T] U6). [score:1]
Promoter region of miR-31 is marked by histone H3 lysine 27 trimethylation (H3K27me3). [score:1]
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[+] score: 227
Other miRNAs from this paper: hsa-mir-200a
Selective targeting of DLL3 or ACOT7 recapitulates miR-31 and/or miR-200a -mediated suppression of Y79 cell proliferationTo determine if selective reduction of endogenous DLL3, PPP6C, STK40, or ACOT7 could recapitulate the inhibitory effect on proliferation mediated by miRNAs-31 and/or -200a, we transiently introduced small interfering RNAs (siRNAs) that are designed to target specific mRNAs for degradation (Fig 7). [score:9]
mirVana [TM] miRNA inhibitor transfectionsHuman retinoblastoma cell lines (Y79 and Weri1) were reverse transfected using RNAiMAX (Invitrogen) in triplicate with 30 picomoles of negative mirVana [TM] mimic inhibitor (Ambion, Catalog #:4464076), miRNA-31-5p inhibitor (Ambion, Catalog #:4464084, ID: MH11465), miRNA-200a-3p inhibitor (Ambion, Catalog #:4464084, ID:MH10991), or “Mix”. [score:9]
Our findings collectively suggest that contextually dependent loss of miRNA-31 and miRNA-200a expression promotes retinoblastoma progression through selective downregulation of targets associated with rapid proliferation. [score:8]
In order to elucidate the genetic mechanisms underlying our observations, we used a gene expression array (>47,000 probes) to identify those genes most differentially expressed after increasing miRNA-31 or miRNA-200a expression individually, or when co -overexpressed together (Mix) in Y79 cells, as compared to controls. [score:8]
Among the ten most significantly downregulated genes following miRNA-31 overexpression, we identified targets that could be important for retinoblastoma progression (Table 1 and S1 Data). [score:8]
MicroRNA-target gene-prediction analyses indicate that miRNA-31 and miRNA-200a could significantly target genes that are expressed in primary retinoblastomas, such as TIAM1 [29] (S2 Table). [score:7]
Human retinoblastoma cell lines (Y79 and Weri1) were reverse transfected using RNAiMAX (Invitrogen) in triplicate with 30 picomoles of negative mirVana [TM] mimic inhibitor (Ambion, Catalog #:4464076), miRNA-31-5p inhibitor (Ambion, Catalog #:4464084, ID: MH11465), miRNA-200a-3p inhibitor (Ambion, Catalog #:4464084, ID:MH10991), or “Mix”. [score:7]
We further noted downregulation of protein phosphatase 6 catalytic subunit (PPP6C), whose encoding protein is significantly correlated with malignant mesothelioma proliferation and also previously validated as a direct target of miRNA-31 [35]. [score:7]
These include serine/threonine kinase 40 (STK40), which encodes an inflammatory regulator (mediated by suppression of NF-κB), previously shown to be a direct target of miRNA-31 [33]. [score:7]
Most significantly downregulated genes after increased microRNA-31 and -200a expression in Y79 retinoblastoma cells. [score:6]
We did not observe a statistically significant knockdown of DLL3 in Weri1 cells, in contrast to Y79-miR-31 or Y79-miR-200a -expressing cells (Fig 3A and 3B), suggesting a miRNA-target interaction unique to Y79 cells. [score:6]
We also observed downregulation of DLL3 following overexpression of miRNA-31 or miRNA-200a. [score:6]
Top 10 genes downregulated after increased microRNA-31 and -200a expression in Y79 retinoblastoma cells. [score:6]
Conversely, we expressed miRNA inhibitors to evaluate if exerting a greater loss of miR-31 and miR-200a expression had any consequence in their ability to proliferate (S3 Fig). [score:5]
Selective targeting of DLL3 or ACOT7 recapitulates miR-31 and/or miR-200a -mediated suppression of Y79 cell proliferation. [score:5]
Using GOmir, we obtained 305 putative miRNA-31 mRNA targets and 656 putative miRNA-200a mRNA targets (S2 Table). [score:5]
Furthermore, our work is the first to demonstrate that overexpression of miRNA-31 and/or miR-200a results in differential gene expression patterns of ACOT7, DLL3, PPP6C, and STK40 between two phenotypically different retinoblastoma cell lines (Fig 3). [score:5]
Although GOmir only reports T-cell lymphoma invasion and metastasis 1 (TIAM1) as a miRNA-200a target, it was previously validated as a miRNA-31 target in colon cancer cells [30]. [score:5]
In silico analysis to identify pathways involving miRNA-31 and miRNA-200a regulationGOmir [23] was used to identify miRNA-31 and miRNA-200a predicted target genes. [score:4]
In comparison to three normal pediatric retinas, both miRNA-31 and miRNA-200a were significantly downregulated in both cell lines. [score:4]
Based upon previous miRNA profiling reports [12, 16], we hypothesized that downregulation of miRNA-31 and miRNA-200a may be an important contributing factor for retinoblastoma proliferation. [score:4]
0138366.g003 Fig 3(A- C) Expression of ACOT7, DLL3, PPP6C, and STK40 mRNAs as measured by TaqMan qRT-PCR in human retinoblastoma cells (Y79 and Weri1) after transient overexpression of miR-31 (A), miR-200a (B), and co -transfected miRs-31 and -200a, “Mix” (C) as compared to negative control miRNA overexpressing cells. [score:4]
MiRNA-31 did not significantly reduce expression levels of ACOT7, as it is not predicted to be among its targets (Fig 3A and S2 Table). [score:4]
S2 Fig Expression of miRNAs-31 (A,C) and -200a (B,D) as measured by TaqMan qRT-PCR in human retinoblastoma cells (Y79 and Weri1) after transient overexpression of miR-31, miR-200a, or co -transfected miRNAs-31 and -200a (Mix), as compared to negative control miRNA overexpressing cells. [score:4]
In order to overcome obstacles to their long-term survival, we hypothesized that some retinoblastomas may rely upon significantly reducing miRNA-31 and miRNA-200a expression. [score:3]
This is a notable observation because miRNA-31 exhibits tumor suppressive effects in cancer mo dels, including the ovary [13], the pancreas [14], and the brain [15]. [score:3]
Predicted targets of miRNA-31 and miRNA-200a identified by GOmir. [score:3]
Predicted targets of miRNA-31 suggests it has a role in cell trafficking, as it is most significantly enriched in pathways important for clathrin-coated vesicle formation, in addition to cytoskeleton remo deling (S1 Table). [score:3]
We observed no reduction of DLL3, ACOT7, PPP6C, and STK40 protein levels following miRNA-31 and/or -200a overexpression. [score:3]
“Mix” is a 1:1 combination of miRNA-31 and miRNA-200a inhibitor per 5.0E4 cells/well in a 24-well plate. [score:3]
Among the four candidate mRNAs examined, STK40 was the single mRNA significantly reduced in Weri1 cells following miR-31 overexpression. [score:3]
By overexpressing miRNA-31 and -200a in vitro, we demonstrated that miRNA-31 and/or miRNA-200a significantly reduce Y79 cell proliferation, but not Weri1 cell proliferation (Fig 2). [score:3]
S3 Fig Bar demonstrates percentage in total cells per mL for Y79 (A) and Weri1 (C) at 96 hours post-transfection with indicated miRNA inhibitors for miRNA-31 and/or -200a. [score:3]
We next evaluated the extent to which overexpression of miRNA-31, miRNA-200a, or concurrent overexpression of both miRNAs affects retinoblastoma cell proliferation in vitro. [score:3]
Real-time PCR of miRNA-31 and/or -200a expression following miRNA mimic transfection in Y79 and Weri1 cells. [score:3]
Our early in silico analyses indicated that miRNA-31 and -200a are each capable of regulating pathways important for development, proliferation, apoptosis, cell cycle, and cell adhesion (S1 Table). [score:3]
Retinoblastomas that are regulated by distinct miRNAs, such as miRNA-31 and miRNA- 200a, may enable the development of phenotypic differences in their ability to proliferate and invade local and distant structures, such as the differences that exist between Weri1 and Y79 cells. [score:3]
Using annexin V and 7-AAD as markers, we detected a significant increase in levels of total cell apoptosis in Y79 cells after miR-31 or miR-200a overexpression (Fig 2B). [score:3]
In a profile of 29 human retinoblastomas, miRNA-31 was first reported to be downregulated as compared to 6 normal human retinas [12]. [score:3]
In contrast to our real-time PCR data that demonstrates miRNA-31 can significantly repress STK40 or PPP6C in Y79 cells, we did not observe any difference in STK40 or PPP6C protein expression as compared to controls, which suggests regulatory changes in the abundance of these mRNAs (mediated by miR-31) are inconsequential (S4 Fig). [score:3]
GOmir [23] was used to identify miRNA-31 and miRNA-200a predicted target genes. [score:3]
Table demonstrates the ten most statistically significant pathways enriched for miRNA-31 or -200a targets. [score:3]
For validation of miRNA-31 and miRNA-200a expression in human retinoblastomas, we analyzed a cohort of 21 primary human retinoblastomas. [score:3]
In silico analysis to identify pathways involving miRNA-31 and miRNA-200a regulation. [score:2]
Using real-time PCR, we confirmed that human retinoblastomas exhibit loss of miRNA-31 and -200a expression as compared to normal pediatric retinas (Fig 1). [score:2]
We demonstrate that overexpression of miRNA-31, miRNA-200a, or both miRNAs together (Mix) significantly reduce Y79 retinoblastoma proliferation as compared to control -treated cells by 23.94%, 31.13%, and 24.84%, respectively, at 96 hours post-transfection (Fig 2A). [score:2]
Validation of STK40, PPP6C, and DLL3 knockdown by miRNA-31, miRNA-200a, or miRNA-31/-200a (Mix) was confirmed using real-time PCR in Y79 cells (Fig 3). [score:2]
Target mRNA expression, as measured in log10, in Y79 and Weri1 retinoblastoma cells after expressing miR-31, miR-200a, or “Mix” (miR-31/-200a), as compared to control treated cells were measured for 4 genes (ACOT7, DLL3, PPP6C, STK40). [score:2]
We sought to elucidate the roles of miRNA-31 and miRNA-200a in retinoblastoma proliferation and apoptosis. [score:1]
For retinoblastoma studies, the potential for miRNA-31 and/or -200a to restrict the growth of this rapidly growing tumor and, by consequence reduce its likelihood to invade ocular structures, is critical because evidence of optic nerve invasion is an important feature with adverse prognostic value for the patient [3]. [score:1]
S4 Fig Immunofluorescence staining of PPP6C (A) and STK40 (B) in Y79 cells transfected with a negative miRNA (control), miRNA-31, miRNA-200a, and miR-31/-200a (Mix). [score:1]
Immunofluorescence staining of DLL3 (A) and ACOT7 (B) in Weri1 cells transfected with a negative miRNA (control), miRNA-31, miRNA-200a, and miR-31/-200a (Mix). [score:1]
S5 Fig Immunofluorescence staining of PPP6C (A) and STK40 (B) in Weri1 cells transfected with a negative miRNA (control), miRNA-31, miRNA-200a, and miR-31/-200a (Mix). [score:1]
Human retinoblastoma cell lines (Y79 and Weri1) were reverse transfected using RNAiMAX (Invitrogen) in triplicate with 30 picomoles of a negative mirVana [TM] mimic (Ambion, Catalog #: 4464058), or, miRNA-31 mimic (Ambion, Catalog #: 4464066, ID: MC11465), or miRNA-200a (Ambion, Catalog #: 4464066, ID: MC10991), or, “Mix” where the “Mix” is a 1:1 combination of miRNA-31 mimic and miRNA-200a mimic per 5.0E4 cells/well in a 24-well plate. [score:1]
Western blot analysis from one experiment of PPP6C (D) and STK40 (F) in Y79 cells transfected with a negative miRNA (control), miRNA-31, miRNA-200a, and miR-31/-200a (Mix). [score:1]
from one experiment of PPP6C (D) and STK40 (F) in Y79 cells transfected with a negative miRNA (control), miRNA-31, miRNA-200a, and miR-31/-200a (Mix). [score:1]
0138366.g005 Fig 5 Immunofluorescence staining of DLL3 (A) and ACOT7 (B) in Y79 cells transfected with a negative miRNA (control), miRNA-31, miRNA-200a, and miR-31/-200a (Mix). [score:1]
0138366.g006 Fig 6 Immunofluorescence staining of DLL3 (A) and ACOT7 (B) in Weri1 cells transfected with a negative miRNA (control), miRNA-31, miRNA-200a, and miR-31/-200a (Mix). [score:1]
Western blots of DLL3 (D) and ACOT7 (F) of Weri1 cells transfected with a negative miRNA (control), miRNA-31, miRNA-200a, and miR-31/-200a (Mix). [score:1]
Next, we evaluated the expression of miRNA-31 and miRNA-200a in two human retinoblastoma cell lines, Y79 and Weri1 (Fig 1C). [score:1]
For quantification of immunofluorescence intensity, four independent images from two independent experiments were obtained for each condition (Negative miR, miR-31, miR-200a, Mix). [score:1]
Altogether, our real-time PCR studies coupled with our analyses in silico supports our hypothesis that miRNA-31 and miRNA-200a have an important role in retinoblastoma proliferation. [score:1]
Immunofluorescence staining of DLL3 (A) and ACOT7 (B) in Y79 cells transfected with a negative miRNA (control), miRNA-31, miRNA-200a, and miR-31/-200a (Mix). [score:1]
of DLL3 (D) and ACOT7 (F) in Y79 cells transfected with a negative miRNA (control), miRNA-31, miRNA-200a, and miR-31/-200a (Mix). [score:1]
mirVana [TM] mimic transfectionsHuman retinoblastoma cell lines (Y79 and Weri1) were reverse transfected using RNAiMAX (Invitrogen) in triplicate with 30 picomoles of a negative mirVana [TM] mimic (Ambion, Catalog #: 4464058), or, miRNA-31 mimic (Ambion, Catalog #: 4464066, ID: MC11465), or miRNA-200a (Ambion, Catalog #: 4464066, ID: MC10991), or, “Mix” where the “Mix” is a 1:1 combination of miRNA-31 mimic and miRNA-200a mimic per 5.0E4 cells/well in a 24-well plate. [score:1]
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[+] score: 217
Meanwhile, co-treatment with HIF1AN siRNA and miRNA-31 inhibitor significantly downregulated the level of HIF1AN than miRNA-31 inhibitor treated cells, while the expression level of protein VEGF increased (Figure 7). [score:10]
For example, Asangani et al. found miRNA-31 was downregulated in melanoma which perhaps act as an outcome of DNA methylation and histone methylation mediated by EZH2; miRNA-31 could inhibit the migration and invasion of melanoma cells, it might play a tumor suppressor role by inhibiting oncogenes SRC, NIK, RAB27a, MET [25]. [score:10]
Based on these results, our study suggests that upregulation of miRNA-31 inhibits HIF1AN expression and activates VEGF in keloid scarring. [score:8]
Downregulation of miRNA-31 decreases proliferation, induces apoptosis and inhibits cell cycle of keloid-derived fibroblasts. [score:6]
The expression levels of HIF1AN, HIF-1α and VEGF were examined by after the downregulation of miRNA-31 and HIF1AN. [score:6]
The expression of miRNA-31 was upregulated in keloid. [score:6]
EdU staining demonstrated that miR-31 inhibitor treatment significantly suppressed DNA synthesis of fibroblasts than negative control treatment, and inhibited proliferation of fibroblasts, as demonstrated by CCK-8 assay. [score:6]
The apoptosis rate was upregulated when fibroblasts were transfected with miRNA-31 inhibitor (Figure 4A, 4B). [score:6]
Keloid-derived fibroblasts were seeded and co -transfected with the above constructs and miRNA-31 expression vector, miRNA-31 inhibitor, control vector or negative control. [score:5]
The result showed miR-31 inhibitor significantly inhibited fibroblasts vitality than negative control (Figure 4A). [score:5]
TargetScan, PicTar and miRanda were respectively used to predict putative target genes of miRNA-31. [score:5]
Luciferase reporter assays were performed to validate whether HIF1AN expression was indeed a direct target of miRNA-31. [score:5]
TargetScan, PicTar and miRanda were used to predict the target genes of miRNA-31. [score:5]
HIF1AN is the target gene of miRNA-31 in keloid-derived fibroblastsThrough the query in the TARGETSCAN database (http://www. [score:5]
Figure 6 (A, B), The expression levels of HIF1AN were examined by after the downregulation of miRNA-31; (C), miRNA-31 binding sites within HIF1AN 3’UTR; (D), Luciferase activity assays with the wild-type 3’UTR or mutated 3’UTR of HIF1AN cotransfeced with vector or miRNA-31. [score:5]
These results suggested that miRNA-31 may regulate the proliferation, apoptosis and cell cycle of keloid-derived fibroblasts via HIF-1α/VEGF signaling pathway by targeting HIF1AN. [score:4]
In this study, through loss-of-function strategies, we found that miRNA-31 could regulate cell proliferation, apoptosis and cell cycle progression of keloid-derived fibroblasts by targeting HIF1AN. [score:4]
Interestingly, two miRNAs, including miRNA-31 and miRNA-214, were detected by 4 studies, indicating that the both miRNAs are the common regulation factor of fibrotic disease, and may play an important role in keloid scarring (Figure 1A, 1B). [score:4]
Inflammatory cytokines can promote miRNA-31 transcription, and miRNA-31 participates in psoriasis inflammation through affecting the secretion of inflammatory cytokines by targeting serine/threonine kinase 40 (STK40) which can negatively regulate NF-kB pathway [22]. [score:4]
These results indicated that HIF1AN was a direct target gene of miRNA-31. [score:4]
miRNA-31 has been found to be dramatically upregulated in psoriatic lesions. [score:4]
Here we confirmed that miRNA-31 was upregulated in keloid tissues and keloid-derived fibroblasts. [score:4]
The expression of miRNA-31 was further validated by qRT-PCR in the skin biopsy samples of 15 keloid patients and 15 healthy controls. [score:3]
Figure 3 (A), GO analysis of miRNA-31 target genes; (B), KEGG pathway analysis. [score:3]
The miRNA-31 mimic or miRNA-31 inhibitor and negative control (NC) were chemically synthesized by Ribobio Co. [score:3]
HIF1AN is the target gene of miRNA-31 in keloid-derived fibroblasts. [score:3]
The result showed there were 114 predicted target genes of miRNA-31 in all of the three programs. [score:3]
Transfection of miRNA-31 mimic and miRNA-31 inhibitor. [score:3]
In this study, we aimed to identify the miRNA expression profiles in keloid and investigate the biological functions of miRNA-31 that may serve as a novel target for prevention and treatment in keloid scarring. [score:3]
In brief, fibroblasts which transfected with miRNA-31 inhibitor, HIF1AN siRNA and normal keloid-derived fibroblasts were seeded at 5×10 [3] cells per well in 96-well plates for triplicate. [score:3]
The results of cell cycle demonstrated that the cellular proportion in G0/G1 phase in miRNA-31 inhibitor group was increased to 78.8% (Figure 5C, 5D). [score:3]
We further found that HIF1AN was a target of miRNA-31. [score:3]
The expression of miRNA-31 in tissues and cells. [score:3]
When keloid-derived fibroblasts grew to 50-70% confluence, miRNA-31 mimic or miRNA-31 inhibitor was transfected and incubated at 37 °C. [score:3]
At the same time, we noted that the expression of miRNA-31 was negatively correlated with the protein level of HIF1AN. [score:3]
However, there are contrary expression and functions of miRNA-31 in other tumors [24]. [score:3]
HIF1AN which is inversely associated with Notch activity could be suppressed by miRNA-31 thus promoting cell differentiation. [score:3]
miR-31 inhibitor and relevant negative control were transfected into keloid-derived fibroblasts. [score:3]
To explore the possible signaling pathways involving miRNA-31 and HIF1AN in keloid-derived fibroblasts, HIF-1α and VEGF expression level were detected. [score:3]
Wang et al. found that miRNA-31 was also overexpressed in cutaneous squamous cell carcinoma (cSCC) and promoting effects of miRNA-31 in cell migration, invasion and colony formation of cSCC indicated the oncogenic role of miRNA-31 in cSCC [23]. [score:3]
In addition, we also explored the expression of miRNA-31 in keloid-derived fibroblasts and normal human fibroblasts, which was consistent with our combined analysis of previous microarray results (Figure 2B). [score:3]
In the present study, we demonstrated that miRNA-31 functions in the development of keloid scarring by negative regulation of the major components in the cell proliferation and cell apoptosis. [score:3]
The forward and reverse segment including miR-31 -binding sites in 3’-untranslated region (3’-UTR) of HIF1AN was synthesized by Ribobio Co. [score:3]
Results showed that miRNA-31 inhibited luciferase activity in fibroblasts with the wild-type HIF1AN 3’-UTR reporter plasmid carried (Figure 6D). [score:3]
Further studies demonstrated that miRNA-31 was related to keratinocytes proliferation and differentiation, miRNA-31 transcription could be induced by NF-kB and thus promoted cell proliferation via inhibiting protein phosphatase 6 (ppp6c) [21]. [score:3]
Keloid-derived fibroblasts which transfected with miRNA-31 inhibitor, HIF1AN siRNA and normal fibroblasts were seeded at 96-well plates 1×10 [4] cells per well and incube 24 hours. [score:3]
On the basis of previous miRNA microarray studies, we screened another miRNA, miRNA-31, that may have important regulatory potential in keloid. [score:2]
Our results demonstrated that VEGF participated in regulation process of miRNA-31/HIF1AN on biological function of fibroblasts. [score:2]
Indicating that miRNA-31 directly binds to HIF1AN 3’-UTR at the predicted binding sites. [score:2]
Taken together, our findings indicate that miRNA-31 can induce the cell proliferation of keloid-derived fibroblasts by down -regulating HIF1AN. [score:2]
We obtained the sequence of the mature miRNA-31-5p from miRBase (http://www. [score:1]
Bioinformatics analysis and functional prediction of miRNA-31. [score:1]
Bioinformatic analysis and functional prediction of miRNA-31. [score:1]
The results showed that the level of miRNA-31 was increased more than 3 folds (Figure 2A). [score:1]
Chen and Peng et al suggested miRNA-31/HIF1AN nexus altered cell proliferation, migration, and invasion in colorectal cancer cell lines and contributed to keratinocyte differentiation [24, 29]. [score:1]
Figure 9Effects of miRNA-31 siRNA and HIF1AN siRNA on (A, B), apoptosis and (C, D), cell cycle of fibroblasts. [score:1]
We consider that miRNA-31/HIF1AN/VEGF pathway may play a critical role in the process of keloid. [score:1]
Figure 5Effect of miRNA-31 on (A, B), apoptosis and (C), cell cycle of fibroblasts. [score:1]
org, Version 7.1), we predicted that there was a consequential pairing between miRNA-31 and binding sites in the 3’ UTR of the gene HIF1AN (Figure 6C). [score:1]
In order to investigate whether miRNA-31 can affect the expression of HIF1AN, we extracted the total protein of fibroblasts and carried out the. [score:1]
Schematic of the described interaction between miRNA-31, HIF1AN and VEGF. [score:1]
Therefore, this miRNA-31/HIF1AN nexus in keloid scarring agrees with previous studies [24, 29]. [score:1]
The effect of miR-31 on S phase of cell cycle progression was verified using (Figure 4B, 4C). [score:1]
Manipulation of miRNA-31 may represent a novel therapeutic strategy for treating the keloid scarring. [score:1]
However, so far, the possible roles of miRNA-31 in this pathogenic process are still unclear. [score:1]
miRNA-31 and HIF1AN were further quantitated by qRT-PCR using an ABI7500 Real-Time PCR System in tissues and fibroblasts. [score:1]
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[+] score: 213
During the medulloblastoma tumorigenesis process, genetic or epigenetic inactivating mechanisms cause a shutdown of miR-31 expression, which leads to an enhanced expression of its downstream target gene MCM2. [score:7]
To determine if down-regulation of miR-31 is a general phenomenon associated with tumorigenesis, we examined miR-31 expression in two established lines of human medulloblastoma cells DAOY and D283, and a number of lung, breast, liver, prostate, and soft tissue cancer lines including A549, NCI-H322M, MCF-7, MDA-MB-231, HEPG2, HEP3B, PC-3, DU-145, RD, and RH30. [score:6]
In our study, a significant down-regulation of MCM2 transcript and protein was observed after miR-31 overexpression in DAOY cells. [score:6]
Our data showed that expression of MCM6, the only MCM complex subunit that possesses miR-31 recognition sites in its 3'-UTR, was not changed after miR-31 overexpression. [score:5]
To validate the NanoString data, we examined miR-31 expression in an enlarged series of normal cerebellar (n=9) and medulloblastoma tissues (n=8), and found that miR-31 was indeed down-regulated in all medulloblastoma samples compared to the normal controls (Figure 1C). [score:5]
Indeed, our results indicated that restoring miR-31 expression strongly suppresses the ability of DAOY cell to grow, form anchorage dependent colonies in vitro, as well as xenograft tumors in nude mice. [score:5]
DAOY cells lack a functional TP53 [34], but our data showed that restoring miR-31 expression is sufficient to inhibit their growth. [score:5]
Putative targets of miR-31 predicted by MiRanda, Targetscan and PITA. [score:5]
We examined the levels of chromatin-bound MCM2 in miR-31 expressing DAOY cells using immunoblotting analysis and immunofluorescence staining, and found that restoring miR-31 expression caused a marked decrease in the levels of chromatin-bound and nuclear matrix-bound MCM2, but those of the cytosolic fractions were not affected at all. [score:5]
Our results indicate that miR-31 also suppresses tumor initiation by targeting minichromosome maintenance complex component 2 (MCM2), thus providing a check point control over initiation of DNA replication. [score:5]
Moreover, there was no difference in expression levels of other MCMs between miR-31 -overexpressing cells and control cells (data not shown). [score:5]
The expression level in vector control cells was set to 1. (C) Immunoblot analysis of MCM2 protein levels in vector control and two independent pools of miR-31 expressing DAOY cells. [score:5]
To determine if miR-31 indeed possesses a tumor suppressor function, we generated a pool of stable DAOY cells, P-miR31 cells, carrying genomically integrated copies of MSCV transcription units that constitutively express miR-31 (Figure 2A). [score:5]
In contrast, quantification of the average MCM2 immunofluorescence of the nucleus revealed a significant difference between control and miR-31 expressing cell nuclei at the G1/S transition, with the mean value of miR-31 expressing cells nuclei being much lower than that of vector carrying cells nuclei, especially in the early phases of stimulation (Figure 4F, right panel). [score:5]
In the present study, we describe that miR-31 is significantly under-expressed in the Ptch [+/-] mouse medulloblastoma mo del and human medulloblastoma cell lines, suggesting that aberrant miR-31 expression might be a common event in the medulloblastoma tumorigenesis. [score:5]
Through bioinformatics and expression analysis, we identified MCM2 as a putative target of miR-31. [score:5]
Restoring miR-31 expression inhibits DAOY cell growth, colony formation and xenograft tumorigenesis. [score:5]
Six of this group contain miR-31 recognition sequences in their 3'-UTR, but RT-PCR experiments indicated that only MCM2 was down-regulated in the P-miR-31 DAOY cells (Figure 3B and Table 1). [score:4]
Down-regulation of miR-31 in mouse and human medulloblastoma cells. [score:4]
MCM2 is a direct target gene of miR-31. [score:4]
Thus, restoration of miR-31 expression in DAOY cells has an equivalent impact on DNA synthesis as knockdown of MCM2. [score:4]
Mutations in the two miR-31 target sites are underlined. [score:4]
We also investigated whether regulation of MCM2 expression by miR-31 influences the expression of other members of the MCM complex. [score:4]
Thus, MCM2 is a direct target of miR-31. [score:4]
Figure 2 (A) DAOY cells were transfected with constitutive miR-31 expression vector, and subsequently selected in puromycin. [score:3]
Following this procedure, we found that the amounts of the soluablized chromatin-bound MCM2 in the S2 fraction and the nuclear matrix associated MCM2 in the P2 fraction in P-miR-31 DAOY cells were significantly lower than those in the control MSCV DAOY cells (Figure 4E), indicating that miR-31 re -expression affects the effective concentration of MCM2 in the chromatin. [score:3]
DAOY cells carrying constitutive miR-31 expressing or control vectors were co -transfected in 24-well plate with 500 ng pGl3-control vector (pGl3-ck) or pGl3-MCM2-wt-3'UTR or mutant reporter plasmids, and 20 ng of pRL-TK plasmid (Renilla) using Lipofectamine 2000. [score:3]
Western analysis also showed marked reduction of MCM2 protein level in the two independent pools of miR-31 expressing DAOY cells (Figure 3C). [score:3]
A mo del for the role of miR-31 in suppressing medulloblastoma tumorigenesisi. [score:3]
Statistical analysis for tumor volumes from control (MSCV) and miR-31 expressing group (P-miR-31) at different time point was shown on right panel. [score:3]
Twenty mice were injected subcutaneously with either 3 × 10 [6] control vector (ten animals) or miR-31 expressing DAOY cells (ten animals). [score:3]
In the human genome, miR-31 is located at 9p21.3, in the vicinity of tumor suppressor, p16 [CDKN2A], which is frequently deleted in solid tumor cells [29, 30]. [score:3]
In vivo xenograft studyTwenty mice were injected subcutaneously with either 3 × 10 [6] control vector (ten animals) or miR-31 expressing DAOY cells (ten animals). [score:3]
It is possible that miR-31 was deleted fortuitously along with the p16 [CDKN2A] locus, or alternatively, miR-31 may have a tumor suppressor function of its own. [score:3]
Here, we report that miR-31, selected from our deep sequencing analysis of the spontaneous Ptch [+/-] mouse mo dels, is a potent inhibitor of medulloblastoma cell growth. [score:3]
The results indicated that except for A549, RD, PC-3 and DU-145, expression of miR-31 was completely abolished in the rest group (Figure 1D), suggesting that miR-31 might impose a hindrance to cell growth that must be overridden during tumorigenesis. [score:3]
Since DAOY cells are TP53 negative, the tumor suppressor function of miR-31 is likely mediated by mechanisms other than apoptosis. [score:3]
These new data suggest that miR-31 may possess tumor suppressor functions of its own. [score:3]
MCM2 is a target of miR-31. [score:3]
Because overexpression of miR-31 showed decreased proliferation and G1/S delay, it is probable that the reduced chromatin-bound MCM2 levels are entirely attributable to growth defects. [score:3]
When attached to the end of a luciferase reporter, the MCM2 3'-UTR repressed the luciferase activity in the P-miR-31 DAOY cells but not in the control MSCV DAOY cells (Figure 3E), presumably due to microRNA -mediated translational repression or mRNA degradation. [score:3]
Stabe DAOY cells overexpressing miR-31 were synchronized at the G0/G1 boundary by serum deprivation and thereafter were released into fresh medium containing 10% serum. [score:3]
MiR-31 is down-regulated in mouse and human medulloblastoma cells. [score:3]
DAOY cells stably expressing miR-31 were fractionated into Triton-soluble (S1 fraction) and –insoluble fractions by CSK/0.5% Triton X-100 buffer. [score:3]
Right panel shows the average MCM2 immunofluorescence per nucleus of vector control and miR-31 expressing DAOY cell nuclei at the G1/S transition. [score:3]
One of these microRNAs is miR-31 that has been shown previously to play an important role in metastasis [22, 24], and also reported as being down-regulated in human medulloblastomas compared to normal cerebella [28]. [score:3]
Thus, our experiments demonstrated that miR-31 indeed possesses the ability to suppress medulloblastoma tumor cell growth both in vitro and in vivo. [score:3]
Consistent with previous reports [33], chromatin association of MCM2 increased dramatically during G1/S in both vector control and miR-31 expressing DAOY cells (Figure 4F). [score:3]
MiR-31 has emerged as an important player in a number of cancers acting as a potent suppressor of proliferation in ovarian, prostate and breast cancer cells [22- 24]. [score:2]
During the process, however, the rate of MCM accumulation in miR-31 expressing DAOY cells was markedly retarded compared to that of the vector control cells. [score:2]
Intriguingly, we found that, like that of MCM2, the level of chromatin-bound and nuclear matrix-bound MCM7 was also reduced in miR-31 expressing DAOY cells compared to the vector control cells (data not shown). [score:2]
To determine if miR-31 regulates MCM2 function, we compared the effects of MCM2 knockdown and miR-31 re -expression on the ability of DAOY cells to incorporate fluorescence dye, EdU, which is a measure of DNA synthesis. [score:2]
The wild type luciferase reporter was generated with a DNA fragment covering two putative miR-31 binding sites. [score:1]
In the present report, we performed genomic PCR detection of regions surrounding the p16 [CDKN2A] locus and found that miR-31 was deleted along with p16 [CDKN2A] in DAOY cells. [score:1]
It is possible that the deletion of miR-31 in DAOY cells was consequential to the deletion of p16 [CDKN2A], which is a frequent event associated with immortalization. [score:1]
To detect the expression of mature miR-31 in different cancer cells, the RT product was amplified in conventional PCR using a miR-31-specific forward primer and the universal reverse primer. [score:1]
To test this, we assessed the levels of chromatin-bound MCM2 in P-miR-31 DAOY cells during G1/S. [score:1]
FACs analysis indicated that 50.43% of P-miR-31 DAOY cells accumulated in the G1 phase, comparing to 40.43% of MSCV DAOY cells, and the percentage of P-miR-31 DAOY cells in the S phase was reduced correspondingly to 44.80% from 53.10% of the control cells (Figure 2C). [score:1]
Toward that end, we also identified Gli consensus DNA -binding sequences in the promoter region of miR-31 gene, suggesting a potent connection between miR-31 and Shh pathway in medulloblastoma. [score:1]
MiR-31 regulates MCM2 function at the DNA replication origin. [score:1]
On the basis of our results, we propose a mo del for the role of miR-31 in medulloblastoma (Figure 5). [score:1]
The 3'-UTR of MCM2 contains two closely situated putative miR-31 recognition sequences (Figure 3D). [score:1]
However, several recent reports showed that the miR-31 promoter was epigenetically inactivated through DNA hypermethylation in CpG islands in primary breast and prostate cancer tissues, independent of the status of the p16 [CDKN2A] locus[36, 37]. [score:1]
Relative to those carrying the empty MSCV viral vector, the MSCV DAOY cells, P-miR-31 DAOY cells grew much slower by daily cell counts (data not shown) and had reduced rate of metabolism as evident by quantification using tetrazolium dye, 3-(4,5-dimethylthiazol -2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Figure 2B). [score:1]
Concurrent deletion of both miR-31 and p16 [CDKN2A] was observed previously in multiple melanomas and urothelial carcinomas in the bladder [29, 30]. [score:1]
of DNA isolated from DAOY cells indicated a large region at the p16 [CDKN2A] locus extending at approximately 1 Mbps both up and down stream, including the miR-31 gene (Figure 1E). [score:1]
These data suggest that reintroducing miR-31 likely restored a G1 to S phase check point control. [score:1]
The miR-31 gene is located within the first intron of a previously uncharacterized gene, LOC554202, at 9p21.3, adjacent to tumor suppressor p16 [CDKN2A]. [score:1]
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Since its expression level is increased in head and neck cancer, hepatocellular carcinoma and colorectal cancer, but decreased in breast cancer, gastric cancer and prostate carcinoma, it was suggested that miR-31 can behave as either a tumor suppressor or an oncogenic miRNA, depending on the transcriptional context in which it is expressed and influenced several cancer associated phenotypes including cell growth, cell migration, metastasis and the sensitivity to chemotherapeutic agents as well [6], [16], [17]. [score:7]
Interestingly, 4 miRNAs were found to be up-regulated in cSCC and among these, miR-31 was the most up-regulated [7]. [score:7]
Recently, we observed the deregulation of miRNA expression in cSCC and identified miR-31 as the most up-regulated miRNA in cSCC compared with healthy skin as determined by miRNA array [7]. [score:6]
To examine whether overexpression of miR-31 in cSCC is due to its up-regulation in tumor cells, or the altered cellular composition of the tumor e. g. due to infiltration of immune cells, we performed in situ hybridization on healthy skin (n = 6) and cSCC (n = 5) specimens. [score:6]
In this study we demonstrate the overexpression of miR-31 in cSCC, specifically in tumor cells, excluding the possibility of cellular infiltration as an explanation for miR-31 overexpression in whole-tumor samples. [score:5]
In conclusion, our results demonstrate that high endogenous level miR-31 in cSCC cells promote their migration, invasion and colony forming ability, and that inhibition of miR-31 can suppress these phenotypes. [score:5]
To investigate the role of miR-31 in cSCCs cells, we overexpressed miR-31 or inhibited endogenous miR-31 in a human metastatic cutaneous SCC cell line, UT-SCC-7, by transfecting miR-31 precursors (Pre-miR-31) or inhibitors (Anti-miR-31) respectively. [score:5]
Several important genes in which regulate cell movement and cytoskeletal have been reported to be direct target of miR-31 including ITGA5, RDX and WAVE3 [21], [22]. [score:5]
Interestingly, miR-31 was not overexpressed in AK compared to healthy skin, suggesting that the up-regulation of miR-31 occurs late during tumorigenesis when lesions become invasive. [score:5]
For functional studies, cells were transfected with 10 nM miR-31 miRNA precursor (Pre-miR-31) or miRNA precursor negative control #1 (Scramble) (Ambion) and with 10 nM miRIDIAN miR-31 hairpin inhibitor or miRNA hairpin inhibitor negative control #1 (Scramble) (ThermoFisher Scientific, Västra Frölunda, Sweden) using Lipofectamine 2000 (Life Technologies, Stockholm, Sweden) following the manufacturers' instruction. [score:5]
Similarly, qRT-PCR analysis of miR-31 expression in an independent set of formalin-fixed, paraffin embedded (FFPE) samples, including 13 healthy skin biopsies and 55 cSCCs showed significant overexpression of miR-31 in cSCC (p<0.001, Figure 1B). [score:5]
Overexpression and inhibition of the mature, biologically active form of miR-31 was confirmed by real-time PCR after transfection (Figure 2). [score:5]
Overexpression and inhibition of the mature, biologically active form of miR-31 was confirmed by real-time PCR after transfection. [score:5]
Supporting our findings, the overexpression of miR-31 in cSCC was previously observed reported by Sand, et al who performed miRNA expression profiling in cSCC tissues [18]. [score:5]
Although miR-31 is typically down-regulated in primary breast tumors, high miR-31 levels were detected in plasma samples of breast cancer patients, and also in patients with other cancer types such as oral carcinoma. [score:4]
MiR-31 was highly expressed in cSCC cells but had low expression in healthy epidermal cells (Figure 1C). [score:4]
In this study, we determined that miR-31 is overexpressed in cSCC and regulates several hallmarks of cancer including cell migration, invasion and colony formation. [score:4]
Up-regulation of miR-31 in skin squamous cell carcinoma. [score:4]
In contrast, down-regulation of miR-31 in primary breast tumor was associated with metastasis [23], indicating a complex role for miR-31 in cancer, that could be dependent on cancer type and tumor stage. [score:4]
Our observation that endogenous miR-31 expression promotes the colony forming ability of cSCC cells indicates the importance of miR-31 in the regulation of cSCC survival. [score:4]
This implies that the increase in miR-31 expression is a late event in neoplastic evolution after formation of an invasive lesion. [score:3]
Representative micrographs for miR-31 expression in healthy skin (Healthy) and cSCC tissues (SCC) are shown. [score:3]
MiR-31 expression was normalized between different samples based on the values of U48 small nucleolar RNA expression (Life Technologies, assay ID: 001006). [score:3]
These results indicate the importance of miR-31 in cSCC hallmarks and its potential as a target for cSCC treatment. [score:3]
Transfection efficiency of anti-miR-31 and pre-miR-31 represented as miR-31 expression level. [score:3]
Inhibition of miR-31 in UT-SCC-7 cells strikingly reduced both migration (2-fold decrease, p<0.001) and invasion (5-fold decrease) of UT-SCC-7 cells (Figure 5A) demonstrating that endogenous miR-31 in UT-SCC-7 cells contributes to these processes. [score:3]
Vice versa, overexpression of miR-31 resulted in a significant increase of cell migration (3-fold increase, p<0.05) and invasion (1.6-fold increase, p<0.05) (Figure 5B). [score:3]
In contrast, significant increase of colony formation was observed in UT-SCC-7 cells upon ectopic overexpression of miR-31 (4-fold increase, p<0.001) (Figure 6B). [score:3]
We found that inhibition of miR-31 in UT-SCC-7 cells did not change the rate of cell proliferation, indicating that the effect of miR-31 on wound closure was not due to its effect on cell proliferation (Figure 4). [score:3]
Similar to our findings, the role of miR-31 in stimulating tumor cell migration and metastasis formation was previously reported in colorectal cancer upon inhibition of miR-31 [16]. [score:3]
Interestingly, miR-31 expression was not significantly altered in actinic keratosis, the precancerous lesions which already have substantial genomic instability but which have not became invasive [2]. [score:3]
To validate the array data and to explore the expression of miR-31 during skin cancer progression, we performed quantitative real-time PCR (qRT-PCR) on a set of RNA isolated from fresh-frozen skin biopsies obtained from healthy skin (n = 21), actinic keratosis (AK, pre-cancerous skin lesions, n = 12), and cSCCs (n = 13). [score:3]
UT-SCC-7 cells were transfected with miR-31 inhibitors (anti-miR-31) or control oligonucleotides (scramble) (A); miR-31 precursor RNAs (pre-miR-31) or miRNA precursor control (scramble) (B). [score:3]
The activation of anti-apoptotic and pro-survival cascades by miR-31 has also been reported in colorectal cancer via the repression of RASA1, a suppressor of RAS oncogene [16]. [score:3]
Moreover, overexpression of miR-31 accelerated this process (Figure 3B). [score:3]
We demonstrated that miR-31 is significantly up-regulated in cSCC as compared to healthy skin and AK lesions (p<0.001 for both) (Figure 1A). [score:3]
Moreover, some miRNAs, for example, miR-31, can behave as either tumor suppressor or oncogenic miRNA depending on the tissues they are located [11]. [score:3]
Inhibition of endogenous miR-31 decreased colony forming ability of UT-SCC-7 cells (3-fold decrease, p<0.001) highlighting its role in cell survival (Figure 6A). [score:3]
Inhibition of miR-31 does not affect cSCC proliferation and cell cycle. [score:3]
0103206.g002 Figure 2 (A) UT-SCC-7 cells were transfected with anti-miR-31 or control oligonucleotides (scramble) and the expression level of miR-31 was detected at 24, 48 and 72 h after the transfection by PCR. [score:3]
0103206.g006 Figure 6 UT-SCC-7 cells were transfected with miR-31 inhibitors (anti-miR-31) or control oligonucleotides (scramble) (A); miR-31 precursor RNAs (pre-miR-31) or miRNA precursor control (scramble) (B). [score:3]
MiR-31 is among the most frequently altered miRNAs in human cancers and it has been proposed as a novel molecular target for cancer therapy and chemoprevention [11]. [score:3]
0103206.g005 Figure 5 Representative photomicrographs of transwell results for UT-SCC-7 cells transfected with (A) miR-31 inhibitor (anti-miR-31) or (B) synthetic miR-31 (pre-miR-31) compared to control oligonucleotides (scramble) were taken under x100 original magnification. [score:2]
In order to determine the effect of miR-31 expression on cSCC motility, we have performed scratch assays. [score:2]
Since miR-31 was described as a master regulator of metastasis and has become obvious that it does play a major role in regulating several other cancer associated cellular characteristics as well [13], we set out to determine the potential roles of miR-31 in the regulation of cancer associated phenotypes including cell migration, cell invasion and colony formation of cSCC. [score:2]
MiR-31 is overexpressed in cutaneous squamous cell carcinoma. [score:2]
These results suggest that miR-31 regulates the motility of cSCC cells. [score:2]
Thus, it is not surprising that miR-31 possessed the fundamental role in regulating the invasion-metastasis cascade. [score:2]
Representative photomicrographs of transwell results for UT-SCC-7 cells transfected with (A) miR-31 inhibitor (anti-miR-31) or (B) synthetic miR-31 (pre-miR-31) compared to control oligonucleotides (scramble) were taken under x100 original magnification. [score:2]
These results suggested the involvement of miR-31 in cutaneous squamous cell tumorigenesis. [score:1]
As shown in the representative photographs and calculated wound area in bar charts, inhibition of endogenous miR-31 significantly reduced the rate of wound closure of UT-SCC-7 cells in comparison with control LNA -treated tumor cells (Figure 3A). [score:1]
Scratch-wound assay was performed to assess the migration rate of UT-SCC-7 transfected with (A) miR-31 inhibitors (anti-miR-31) or (B) synthetic miR-31 (pre-miR-31) compared to control oligonucleotides (scramble). [score:1]
These suggested the potential application of miR-31 as a diagnostic marker [24]. [score:1]
Thus, we aimed to determine whether miR-31 can regulate these processes using Transwell migration and invasion assays (Figure 5). [score:1]
Quantification of miR-31 was carried out by TaqMan Real-Time PCR according to the manufacturer’s instructions (Life Technologies, Stockholm, Sweden). [score:1]
0103206.g001 Figure 1 (A) qPCR analysis of miR-31 in healthy skin, actinic keratosis lesion and cSCC in fresh-frozen clinical material. [score:1]
These results demonstrate the role of miR-31 in promoting the migration and invasion of cSCC cells. [score:1]
The enhancement of colony forming ability in UT-SCC-7 by miR-31 indicated the potential roles of miR-31 in cSCC tumorigenesis. [score:1]
Induction of cSCC motility by miR-31. [score:1]
Our results revealed the role of miR-31 in promoting cSCC cells migration and invasion, crucial steps in tumor progression. [score:1]
This is in agreement with previous studies showing an oncogenetic function of miR-31 in lung and oesophageal SCCs, as well as association of miR-31 with poorer relapse prognosis and patient survival in lung and oesophageal SCCs [19], [20]. [score:1]
0103206.g003 Figure 3 Scratch-wound assay was performed to assess the migration rate of UT-SCC-7 transfected with (A) miR-31 inhibitors (anti-miR-31) or (B) synthetic miR-31 (pre-miR-31) compared to control oligonucleotides (scramble). [score:1]
The diagnostic potential of circulating miR-31 in cSCC remains to be determined. [score:1]
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Consistent with the previously mentioned studies, we showed that the forced downregulation of miR-31 significantly inhibited the activity of the RAS-MAPK signaling pathway, possibly through the upregulation of RASA1 expression; however, the downregulation of RASA1 attenuated this change. [score:14]
It was observed that the forced downregulation of miR-31 significantly promoted the protein expression of RASA1, inhibited ICC cell proliferation and enhanced cell apoptosis; however, the forced downregulation of RASA1 expression attenuated these changes, suggesting that RASA1 acted as a downstream effector of miR-31 in ICC cells. [score:13]
Since miRNAs are generally involved in the pathogenesis of cancer by directly regulating the expression of their targets at a post-transcriptional level, we applied bioinformatic methods to predict the potential targets of miR-31. [score:9]
These data suggest that miR-31 promotes GTP binding to RAS by suppressing the expression of RASA1, and further upregulates the activity of the RAS-MAPK signaling pathway by increasing the phosphorylation level of ERK1/2 in ICC cells. [score:8]
Based on the contrasting expression patterns of miR-31 and RASA1, we proposed that miR-31 was involved in the pathogenesis of ICC by directly inhibiting the protein expression of RASA1, a validated oncogene in colorectal, liver and breast cancer, as well as promyelocytic leukemia (12– 16). [score:8]
Following transfection with miR-31 inhibitor, the expression level of miR-31 was significantly reduced (P<0.01; Fig. 2B), while the protein expression of RASA1 showed an increase (P<0.01; Fig. 2C). [score:7]
However, in HCCC-9810 cells cotransfected with miR-31 inhibitor and RASA1 siRNA, the cellular apoptosis rate was not increased compared with that of untreated HCCC-9810 cells, suggesting that miR-31 was able to inhibit cell apoptosis in HCCC-9810 cells, possibly by downregulating RASA1. [score:7]
However, in HCCC-9810 cells cotransfected with miR-31 inhibitor and RASA1 siRNA, the cell proliferation rate was not downregulated, and instead showed a marginal increase when compared with that in the control group, further indicating that RASA1 acted as a downstream effector of miR-31 and was important in the regulation of ICC cell proliferation. [score:6]
These results partially explain why the forced downregulation of miR-31 inhibited cell proliferation and promoted cell apoptosis in ICC cells. [score:6]
In the present study, we showed that the expression of miR-31 was upregulated in ICC tissues. [score:6]
Moreover, these results partially explain why the downregulation of miR-31 inhibits the cellular proliferation and promotes the apoptosis of ICC cells. [score:6]
Six types of commonly used bioinformatic software, including miRanda (17), miRDB, miRWalk (18), RNAhybrid (19), PICTAR5 (20) and Targetscan (21), independently predicted that RASA1 was a direct target of miR-31. [score:6]
Moreover, these observations also suggested that the ability of miR-31 to accelerate cell proliferation was partially via the direct suppression of RASA1 expression. [score:6]
As shown in Fig. 1A, the level of miR-31 expression in ICC tissue was significantly upregulated, when compared with that in normal adjacent tissues (P<0.01). [score:5]
To further confirm these results, we transfected miR-31 inhibitor into HCCC-9810 cells and then examined the expression of miR-31, as well as the protein level of RASA1. [score:5]
Moreover, in HCCC-9810 cells transfected with miR-31 inhibitor, the ratio of phospho-ERK1/2 to total ERK1/2 was markedly decreased, while cotransfection with miR-31 inhibitor and RASA1-specific siRNA also attenuated this change. [score:5]
As shown in Fig. 5, in miR-31 -downregulated HCCC-9810 cells, the cell apoptosis rate was significantly higher than that in untreated HCCC-9810 cells (P<0.01), suggesting that miR-31 negatively regulated cell apoptosis in ICC cells in vitro. [score:5]
As demonstrated in Fig. 6, in HCCC-9810 cells transfected with miR-31 inhibitor, the protein level of the GTP-bound RAS was significantly reduced when compared with that in the control group (P<0.01); however, cotransfection with miR-31 inhibitor and RASA1-specific siRNA attenuated this change. [score:4]
These data identified RASA1 as a direct target of miR-31. [score:4]
Our data revealed RASA1 to be a direct target of miR-31. [score:4]
This indicated that the RASA1 was a direct target of miR-31 in HCCC-9810 cells. [score:4]
These data suggest an inverse correlation between miR-31 and RASA1 expression during the tumorigenesis of ICC. [score:3]
Expression of miR-31 in ICC tissues and HCCC-9810 cells. [score:3]
As shown in Fig. 4, in miR-31 -downregulated HCCC-9810 cells, the cell proliferation rate was significantly decreased when compared with that in the control untreated HCCC-9810 cells (P<0.01), indicating that miR-31 was able to enhance cell proliferation. [score:3]
We further examined the expression of miR-31 in the HCCC-9810 cell line. [score:3]
The cells were subsequently cotransfected with psiCHECK-2-RASA1-3′-UTR or psiCHECK-2-mut RASA1-3′-UTR vector in combination with 100 nM miR-31 or 100 mM miR-31 inhibitor, respectively, using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA). [score:3]
RASA1 is a target of miR-31. [score:3]
In conclusion, the present study provides a novel insight into the regulatory pattern of miRNA-31 and RASA1 in ICC in vitro, suggesting that miRNA-31 and RASA1 may become promising candidates for the development of effective strategies for the treatment of ICC. [score:3]
In ICC, a number of miRNAs, including microRNA-21 (miR-21), miR-31, miR-124 and miR-200c, have been demonstrated to show aberrant expression patterns (8– 10), and these miRNAs may therefore participate in the tumorigenesis of this type of cancer. [score:3]
To study the regulatory mechanism underlying the tumorigenesis of ICC, we investigated the targets of miR-31. [score:2]
For the analysis of miR-31 expression, 2 μg RNA was transcribed to cDNA using a stem-loop reverse transcription (RT) primer and an miRNA reverse transcription kit under the following conditions: 16°C for 30 min, 42°C for 30 min and 85°C for 5 min. [score:2]
However, to date, it has not been elucidated whether RASA1 is important in the development of ICC, and the correlation between RASA1 and miR-31 in ICC cells has yet to be revealed. [score:2]
The present study aimed to study the roles of miR-31 and RASA1 in the regulation of ICC cell proliferation and migration, in addition to elucidating the underlying molecular mechanisms. [score:2]
As shown in Fig. 1B, the level of miR-31 expression in HCCC-9810 cells was also significantly higher when compared with that in the control (P<0.01). [score:2]
However, the exact role of miR-31 in the development of ICC has yet to be elucidated. [score:2]
To preliminarily investigate the role of miR-31 in ICC, we assessed the expression of miR-31 in ICC tissues and HCCC-9810 cells. [score:1]
Roles of miR-31 and RASA1 in HCCC-9810 cell apoptosis. [score:1]
In miR-31 and RASA1-3′-UTR-cotransfected HCCC-9810 cells, the renilla/firefly value of luciferase was notably decreased (P<0.05). [score:1]
miR-31 has been demonstrated to be important in various types of cancer, including hepatocellular, squamous cell, ovarian, prostate and urothelial carcinoma, as well as colon, head and neck, gastric and breast cancer (8, 26– 33). [score:1]
These results suggest that miR-31 may be involved in the pathogenesis of ICC. [score:1]
However, in the miR-31 and RASA1 mutated 3′-UTR-cotransfected HCCC-9810 cells, the renilla/firefly value of luciferase showed no difference from that in the control cells (Fig. 2A). [score:1]
Based on the results of the bioinformatic analysis, we performed a dual-luciferase reporter assay to investigate whether RASA1 was a direct target of miR-31. [score:1]
However, the role of miR-31 in ICC has yet to be elucidated. [score:1]
Molecular mechanism for miR-31 and RASA1 in ICC. [score:1]
Roles of miR-31 and RASA1 in HCCC-9810 cell proliferation. [score:1]
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[+] score: 154
Interestingly, the results also indicate that Sfp53 overexpression in the absence of irradiation is insufficient to inhibit miR-31 expression (Fig.   4d). [score:7]
While over -expression of miR-31 was prominent at the higher (lethal) doses that failed to accumulate p53, silencing p53 expression also resulted in revival of miR-31 expression associated with significant cell death induction, thereby confirming the existence of a ‘radioprotective p53 gateway’ in these mo del radioresistant cells. [score:7]
To finally confirm whether radiation -induced accumulation of Sfp53 at sublethal doses plays any role in cytoprotection against radiation -induced apoptosis and/or downregulation of miR-31, Sfp53 expression was silenced using siRNA starting nearly 4 h before irradiation at the sublethal dose of 200 Gy (Fig.   7a). [score:6]
Importantly, radiation -induced accumulation/expression of Sfp53 showed inverse relationship with the alterations in expression of miR-31, which was recently shown to regulate caspase activation and apoptotic induction in these cells [19]. [score:6]
Figure 7p53-si -RNA induce cell death at sub-lethal doses by inhibiting miR-31 downregulation. [score:6]
Initial results during this study indicated that radiation -induced alterations in miR-31 expression have an inverse dose response with those in Sfp53 expression (Fig.   1a,c). [score:5]
Newfound relation between radiation-responsive p53 and miR-31: hyper-phosphorylated Sfp53 binds near miR-31 locus and suppresses its expression to prevent apoptosis. [score:5]
The miR-31 expression was selectively suppressed at 200 Gy dose, which coincidentally resulted in the maximum accumulation of p53. [score:5]
The expression of miR-31 after antisense transfection was quantified using Real Time PCR after 16–18 h, while p53 expression analysis was performed 24 h post transfection with western blotting. [score:5]
While p53 showed maximal accumulation following irradiation at 200 Gy (Fig.   1a), miR-31 expression was previously reported to be unusually suppressed at this dose that failed to induce caspase-3 activity or cell death [19]. [score:5]
These data thus show an inverse relationship between radiation -induced p53 accumulation and miR-31 expression, the overexpression of latter coinciding with cell death induction as reported earlier [19]. [score:5]
A possible explanation for p53 -mediated miR-31 suppression could be that phosphorylated p53 may bind at the miR-31 locus and may negatively compete with other transcriptional factors required for inducing expression of miR-31. [score:5]
It is further important to note that over -expression of Sfp53 also failed to alter miR-31 expression (Fig.   4d), which indicates that cell death induced by Sfp53 in this case might be executed through different mechanisms than the apoptosis induced by lethal radiation doses. [score:5]
The study shows a rather interesting role of p53 phosphorylation status in the radioresistance of Sf9 cells, which seems to work through highly selective suppression of miR-31 expression at sub lethal doses. [score:5]
Functional analysis strongly indicated that tumor-suppressive miR-31 inhibits proliferation and promotes apoptosis in the ovarian tumor cells. [score:5]
In contrast, downregulation of miR-31 was observed at sub-lethal doses, a response that could be associated with radiation resistance in this mo del system. [score:4]
An isolated study reported that miR-31 is the most strongly downregulated miRNA in serous ovarian tumors as well as in many osteosarcoma and pancreatic carcinoma cell lines. [score:4]
Our findings show that Sfp53 negatively regulates the radiation-inducible overexpression of pro-apoptotic miR-31 in the highly radioresistant Sf9 cells reported recently by our laboratory [19]. [score:4]
This differential or selective binding of p53 at miR-31 locus following sub-lethal irradiation suggests a possible regulation of miR-31 expression by Sfp53 when the latter is in hyper-phosphorylated state. [score:4]
Radiation -induced overexpression of miR-31 was shown to promote Bim/Bax -mediated apoptosis in these cells at lethal doses of ionizing radiation. [score:3]
In order to assess whether there is cause-and-consequence relationship between p53 and miR-31 expression and alterations following irradiation, direct binding of p53 near the miR-31 locus was tested using chromatin immunoprecipitation (ChIP) assay. [score:3]
Figure 6Phospho-Sfp53 binds nearby to Sf-miR-31 locus and selectively repress its expression at sub-lethal doses. [score:3]
RNA -based silencing of Sfp53 following sublethal dose irradiation enhances cell death associated with release of miR-31 suppression. [score:3]
Most importantly, this was also associated with nearly complete reversal of miR-31 repression induced by this sub-lethal dose (Fig.   7c), hence corroborating the presence of a selective radiation -induced Sfp53 gateway withholding miR-31 overexpression and cell death. [score:3]
Figure 4Ectopic overexpression of Sfp53 induces caspase-3 dependent apoptosis in miR-31 independent manner. [score:3]
It should be noted here that the absence of accumulation and/or hyper-phosphorylation of Sfp53 following irradiation at the lethal doses (1000Gy-3000Gy) was associated with significant dose -dependent induction of cell death (Figs  1a, 5a), which also involved dose -dependent miR-31 overexpression [19]. [score:3]
Radiation -induced accumulation and nuclear translocation of Sfp53 corresponds inversely with the alterations in miR-31 expression. [score:3]
Recently, we had reported that miR-31 overexpression mediates radiation -induced apoptosis in these mo del insect cells [19]. [score:3]
The higher (lethal) radiation doses failed to display similar response as Sfp53 accumulation was diminished in a dose -dependent manner, which corresponded very well with the overexpression of miR-31. [score:3]
Moreover, the absence of miR-31 binding site at the 3′UTR of Sfp53 suggests that the Sfp53 expression is not influenced by miR-31 (Data not shown). [score:3]
Interestingly, contradicting data from different studies have shown miR-31 as tumor suppressor/oncogenic miRNA and indicated that its role in carcinogenesis may be cell type dependent 21, 22. [score:3]
Quite importantly, the knockdown of Sfp53 prior to irradiation at 200 Gy released the repression of miR-31 and induced significant cell death; thus demonstrating the existence of a ‘p53 gateway’ with a strong radioprotective role in these cells (Fig.   7). [score:2]
2 μl of cDNA was used to analyze the mRNA/miR-31 expression using gene specific primers in Real Time Thermocycler (Stratagene, USA, MX3005P). [score:2]
Our study shows differential binding of Sfp53 at the miR-31 locus selectively following irradiation at 200 Gy (Fig.   6b), which strongly suggests a direct transcriptional repression of miR-31 by hyper-phosporylated Sfp53. [score:2]
On the other hand, binding of p53 near miR-31 locus was not observed at the lethal dose of 2000Gy. [score:1]
Therefore, subdued miR-31 levels at 200 Gy could be the result of an intrinsic radioprotective mechanism, and indicated a possible relation with Sfp53 response. [score:1]
Binding of p53 around the ~1000–2000 bp region of miR-31 sequence was assessed through ChIP strategy in untreated Sf9 cells as well as cells that were irradiated with sublethal or lethal doses. [score:1]
Since Sf9 cells also have an active miR-31 response during radiation -induced cell death [19], we conducted the present study for exploring possible relationship between Sfp53 and miR-31 response following radiation exposure. [score:1]
To detect p53 binding at miR-31 locus, DNA isolated from p53 immunoprecipitated samples at different radiation doses (200 Gy and 2000 Gy) were used. [score:1]
A recent study from our laboratory has demonstrated mediatory role of miR-31 in radiation -induced cell death in a radioresistant insect cell line (Sf9) that carries numerous structural and functional similarities with mammalian/human cells [19]. [score:1]
While designing this strategy, long flanking sequences around SfmiR-31 were used since the exact location of p53 binding at miR-31 promoter site is not possible to predict in Sf9 cells due to non-availability of its annotated genome sequence. [score:1]
suggests Sfp53 binding selectively only at 200 Gy radiation, while in control condition moderate binding of Sfp53 near to miR-31 sequence was observed. [score:1]
For transfection of AS-miR-31 and siRNA against p53, 0.50 μg of each was used with RNAiFect (Qiagen, USA, Cat No. [score:1]
However, the exact Sfp53 binding site at miR-31 locus in Sf9 is not possible to detect because of the unavailability of complete annotated lepidopteran genome sequence at present. [score:1]
Precipitated DNA was subsequently used for Dot blot to verify whether miR-31 also gets immunoprecipitated with p53. [score:1]
In the immuno-precipitated DNA fragments, the presence of miR-31 sequence was identified using (b) dot blot analysis, taking miR-31 specific biotinylated probes to show p53 binding near to (1000–2000 bp region) miR-31 sequence. [score:1]
Figure 1Radiation induced accumulation and translocation of Sfp53 to the nucleus is not associated with miR-31 dependent cell death. [score:1]
We noticed a reverse pattern in the radiation dose-response of p53 versus miR-31 (Fig.   1). [score:1]
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[+] score: 146
Other miRNAs from this paper: hsa-mir-183, hsa-mir-143, hsa-mir-155, hsa-mir-324
Given the upregulation of miR-31 and miR-155 in biopsies from colonic mucosa and isolated epithelial cells in active UC, while IL13RA1 expression is decreased in active UC (Figure 1, Figure 2 and Figure 4) and the direct -targeting of IL13RA1 mRNA by miR-31 and miR-155 (Figure 3 [14]), we hypothesised that these miRs may directly affect IL13RA1 levels and IL-13 signalling in colonic epithelium. [score:10]
Our data indicate that miR-31 and miR-155 upregulation may contribute to the downregulation of IL13RA1 in inflamed mucosa in UC. [score:7]
As expected, miR-31 and miR-155 were found to be upregulated (Figure 4b) in these cells, consistent with their potential to regulate IL13RA1 expression. [score:7]
As another novel finding we show that miR-31 is upregulated in active UC compared to inactive UC or healthy donors and directly targets the 3′UTR of IL13RA1 mRNA. [score:6]
MicroRNA-31 and miR-155 Reduce IL-13 Signalling by Downregulating IL13Rα1 Expression in Gut Epithelial Cells. [score:6]
Amongst miRs identified to potentially target IL13RA1, we selected a subset previously reported to be dysregulated in inflammatory bowel disease (Figure 2a and Table S1): miR-155-5p [28, 29], miR-31-5p [28, 30], miR-183-5p [28] and miR-324-3p [30]. [score:6]
MicroRNA-31 and miR-155 were able to reduce IL-13 signalling in gut epithelial cells through downregulation of expression of IL13Rα1. [score:6]
Given the direct -targeting of IL13RA1 by both miR-31 (Figure 3) and miR-155 [14], we assessed the expression of IL13RA1 and these miRs in isolated epithelial cells from colonic biopsies comparing healthy controls with unaffected and inflamed mucosa from UC patients (Table 3). [score:6]
Exogenous miR mimics of miR-31 and miR-155 can actively downregulate IL13RA1 expression in gut epithelial cells in vitro but also importantly in ex vivo explant cultures from inflamed colonic mucosa from patients with UC (Figure 5e). [score:6]
Co-transfection of the renilla reporter IL13RA1 3′UTR constructs together with a miR-31 expression vector (or control empty vector) showed that over expression of miR-31 significantly reduced luciferase activity of the WT reporter, while it did not affect the activity of the mutated reporter (Figure 3b). [score:5]
Olaru A. V. Selaru F. M. Mori Y. Vazquez C. David S. Paun B. Cheng Y. Jin Z. Yang J. Agarwal R. Dynamic changes in the expression of microRNA-31 during inflammatory bowel disease -associated neoplastic transformationInflamm. [score:5]
Future work will establish whether miR-31 and miR-155 play also a role in other IL-13 related diseases such as asthma and atopic dermatitis as well as targeting other genes involved in UC. [score:5]
MicroRNA-31 and miR-155 individually and in combination are able to significantly decrease IL13RA1 expression (Figure 5a,b) and IL-13 -dependent responses as reflected by decreased IL-13 -dependent phosphorylation of STAT6 (Figure 5c) as well as mRNA expression of SOCS1 and CCL26 in gut epithelium (Figure 5d). [score:5]
Reverse transcription and real-time PCR analysis showed that expression of SOCS1 and CCL26 mRNA was down regulated by miR-31 and miR-155 separately and in combination (Figure 5d). [score:4]
Our data therefore support a key role for miR-31 and miR-155 in UC via directly targeting of IL13RA1 mRNA. [score:4]
Importantly, this is the first time that miR-31 expression has been shown to be overexpressed in active UC patients compared to inactive UC or healthy donors. [score:4]
3.3. miR-31 Directly Targets IL13RA1 mRNA. [score:4]
com/2073-4425/9/2/85/s1, Figure S1: Western blotting of IL13Rα1 colonic biopsies; Figure S2: Representative Western blotting detection of IL13Rα1 in HT-29 cells transfected with pre-miR-31, pre-miR-155 or a combination of both; Figure S3: Representative Western blotting detection of phospho-STAT6 in HT-29 cells transfected with pre-miR-31, pre-miR-155 or a combination of both (pre-miR-31/155); Figure S4: Effects of miR-31, miR-155 and their combination on the expression of SOCS1 mRNA in an IL-13 -dependent and independent manner in HT-29 cells; Figure S5: IL-4 and IL-13 cytokine expression from colonic biopsies from patients with inactive or active UC compared to normal non-UC controls. [score:4]
These results show, for the first time, that miR-31 directly targets IL13RA1 mRNA, binding to the sequence comprising nucleotides 1158–1165 on its 3′UTR. [score:4]
Our data suggests that increased miR-31 and miR-155 may exert a suppressive role against IL-13 -dependent effects in the pathophysiology of UC by reducing IL13Rα1 levels. [score:3]
Mutation of the binding site of miR-31 was done using QuickChange site directed mutagenesis (Stratagene, San Diego, CA, USA) following the manufacturer’s protocol. [score:3]
As it happens in the lung [35], IL-13 may play a protective role against fibrosis in the gut and thus miR-31 and miR-155 may have a pathological role by targeting IL13RA1. [score:3]
We observed a reduction in the IL-13 effect on SOCS1 and CCL26 mRNA expression when miR mimics were used (Figure 5d), but we did not observe an additive effect of miR-31 and miR-155 in combination. [score:3]
To confirm the effects of miR-31 and miR-155 on IL13RA1 expression in human colonic tissue, we used an ex vivo explant culture system. [score:3]
IL13RA1 Expression Is Reduced in Primary Inflamed Ulcerative Colitis Gut Epithelium with Increased miR-31 and miR-155 Levels. [score:3]
Figure 2b shows that only miR-31-5p and miR-155-5p (hereinafter miR-31 and miR-155, respectively) expression was significantly increased in inflamed UC tissue compared to unaffected samples. [score:2]
Figure 5e shows that miR-31, miR-155 and their combination were able to significantly decrease the expression of IL13RA1 mRNA compared to biopsies transfected with scrambled miR control. [score:2]
In summary, we have described a novel mechanism by which increased levels of miRs in UC, namely miR-31 and miR-155, regulate the IL-13 pathway. [score:2]
Thus, while we have demonstrated that miR-31 and miR-155 can regulate IL-13 signalling, the precise role of these miRs in the pathophysiology of UC will require the elucidation of the impact of IL-13 in the human gut. [score:2]
By performing site directed mutagenesis, we generated a mutant version for the predicted binding site of miR-31, with the aim of abrogating binding (mutant (MUT) in Figure 3a). [score:2]
Our work demonstrates a role for miR-31 and miR-155 in the regulation of IL-13 signalling. [score:2]
In silico prediction [27] indicated that miR-31 directly binds to the 3′UTR of IL13RA1 (Figure 2a and Figure 3a). [score:2]
Transfection of Pre-miR-31 and Pre-miR-155 (individually or combined) significantly reduced the expression of IL13RA1 mRNA and protein compared to Control (Figure 5a,b and Figure S2). [score:2]
The IL-13 -dependent phosphorylation of STAT6 was reduced by miR-31 and miR-155 (Figure 5c and Figure S3) as shown by Western blot analysis. [score:1]
Inflamed colonic tissue from patients with UC (Table 4) was directly transfected with Pre-miR-31 or Pre-miR-155 individually (100 nM) or in combination (50 nM each) and compared to 100nM scrambled miR control -transfected biopsies (Control). [score:1]
We generated a reporter construct (Figure 3a) fusing a renilla luciferase reporter gene to the 3′UTR of IL13RA1 containing the sequence for the predicted binding site of miR-31 (wild type (WT) in Figure 3a). [score:1]
HT-29 cells were transfected with 100 nM Pre-miR™ miR precursors (Negative control#1, miR-31, miR-155 or a combination of 50 nM miR-31 + 50 nM miR-155, Thermo Fisher Scientific) using Interferin (Polyplus, New York, NY, USA) following manufacturer’s instructions. [score:1]
The genomic region encompassing miR-31 was amplified by PCR from genomic DNA (gDNA) using AmpliTaq gold DNA polymerase (Thermo Fisher Scientific), subcloned into pCR2.1 TOPO-TA cloning kit (Thermo Fisher Scientific) and then into pCDNA3.1(-) (Thermo Fisher Scientific). [score:1]
We therefore tested the direct binding of miR-31 to IL13RA1 3′UTR mRNA by employing a dual luciferase reporter assay. [score:1]
The reporter for the 3′UTR of IL13RA1 containing the potential binding site for miR-31 was previously generated [14]. [score:1]
Samples were transferred onto a 96 well plate (U-bottom) in 200 μL of Aqix solution and transfected with 100 nM Pre-miR™ miRNA precursors (Negative control #1, miR-31, miR-155 or a combination of miR-31/155 mix (50 nM each)) using Interferin (Polyplus) following manufacturer’s instructions. [score:1]
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[+] score: 122
Regulation of Fzd9 expression is poorly understood, but if we find that miR-31 does not directly regulate Fzd9, known targets of miR-31 could provide direction for future studies of Fzd9 regulation. [score:10]
Previous studies suggest that iloprost binds with Fzd9 to induce anti-cancer signaling, however, prostacyclin may also generate effects by increasing Fzd9 expression 9. Since our data demonstrated an inverse relationship between Fzd9 and miR-31 expression with exposure to iloprost (comparing Figs 2 and 4 with Fig. 6), we hypothesized that miR-31 may be a link between prostacyclin and Fzd9 expression. [score:7]
Fzd9 expression was inversely related to miR-31 expression in vitro and in vivo and decreased with transfection of a miR-31 mimic regardless of CSC or iloprost exposure. [score:5]
In this mo del, cigarette smoke increases miR-31 expression, which prevents prostacyclin from increasing Fzd9 expression. [score:5]
Fzd9 expression was not increased in currently CSC exposed cells with iloprost (CCSC ilo) (Fig. 4a), suggesting that while mir-31 expression changes with iloprost, it is not the only contributor to Fzd9 repression in the presence of cigarette smoke condensate. [score:5]
Fzd9 expression was inverse to miR-31 expression in these mouse mo dels (Fig. 2). [score:5]
When cigarette smoke exposure is removed, prostacyclin reduces miR-31 expression and Fzd9 expression begins to return (Fig. 7). [score:5]
When cigarette smoke is removed, prostacyclin is able to reduce miR-31 expression, increase Fzd9 expression, and initiate prevention signaling through PPARγ. [score:5]
Fzd9 expression decreased in the cells with increased miR-31 expression (Fig. 6f). [score:5]
A miR-31 transgenic mouse has increased lung hyperplasia, adenoma, and adenocarcinoma and promotes KRAS mediated oncogenesis by directly reducing expression of negative regulators of RAS/MAPK signaling 26. miR-31 and let-7 coordinate to maintain balance in proliferation of lung cancer stem-like cells, suggesting a role for miR-31 in the earliest cellular changes leading to lung cancer 27. [score:5]
Of these three, individual qPCR for expression changes after CSC and iloprost exposure in HBEC confirmed that miR-31 expression increased with 4 weeks of CSC and decreased with 4 weeks of iloprost (Fig. 6a). [score:5]
Iloprost tended to reduce miR-31 expression in both previous (PCSC ilo) and current CSC (CSC ilo) exposure, though it is unclear why miR-31 expression is lower in CCSC ilo compared to PCSC ilo. [score:4]
To determine whether changes in miR-31 affected Fzd9 expression, a miR-31 mimic was transfected into cells with 6 weeks of iloprost treatment. [score:3]
miR-31 mediates prostacyclin and cigarette smoke induced Fzd9 expression changes. [score:3]
miR-31 increased with exposure to cigarette smoke and decreased with lung specific prostacyclin overexpression or iloprost treatment. [score:3]
Evidence for the technical feasibility of miR-31 marker detection in surrogate biospecimens was presented using digital PCR to analyze expression in sputum 29. miR-31 may be useful as a biomarker for monitoring iloprost activity in surrogate specimens as an alternative to biopsy by bronchoscopy during chemoprevention treatment. [score:3]
miR-31 expression induced by mimics was not reduced by the addition of iloprost. [score:3]
In the area of WNT signaling, miR-31 stimulates canonical Wnt/β-catenin by depleting repressors of the pathway and increases expression of non-canonical Wnt5a, which induces EMT 22. [score:3]
We propose a mo del where cigarette smoke exposure decreases Fzd9 expression, in part by increasing miR-31 and preventing normal signaling between prostacyclin and Fzd9. [score:3]
How to cite this article: Tennis, M. A. et al. Prostacyclin reverses the cigarette smoke -induced decrease in pulmonary Frizzled 9 expression through miR-31. [score:3]
miR-31 was slightly lower in previous CSC (PCSC) exposed cells, suggesting the full effect of CSC removal on miR-31 expression required more time without CSC. [score:3]
The presence of high levels of miR-31 prevented the increased Fzd9 expression induced by iloprost, suggesting that miR-31 is a link between iloprost and Fzd9. [score:3]
Further studies are needed to determine whether other WNT pathway components, such as Dkk or SFRP1, are targeted by miR-31 or involved in iloprost stimulated Fzd9 signaling. [score:3]
miR-31 mimic expression that could not be overcome by iloprost also resulted in lower Fzd9 levels (Fig. 6f). [score:3]
miR-31 has many predicted targets, including hub genes PIK3CA, JUN, MAPK1, MAPK3, CCND1, FOS, MDM2, KRAS, EGFR, PTK2, and VEGFA 33. [score:3]
We found that miR-31 expression increased with smoke exposure in the one week mouse mo del and was abrogated with increased prostacyclin levels in PGIStg mice (Fig. 6b). [score:3]
Iloprost increases Fzd9 by reducing miR-31 expression. [score:3]
miR-31 expression increased in both previous (PCSC) and current CSC (CCSC) exposed cells compared to control cells. [score:2]
It is also involved in regulation of lung cancer stem-like cells 27. miR-31 was proposed as a biomarker for drug activity in a murine lung cancer chemoprevention mo del using vinyl carbamate and indole-3-carbinol 28. [score:2]
In whole lung from the PGIStg mice compared to wild type mice, miR-31 expression decreased (Fig. 6c). [score:2]
To examine a potential role for miR-31 in iloprost chemoprevention in former smokers, we measured expression in our 20 week in vitro mo del that exposed HBEC cells to CSC and iloprost (Fig. 6d). [score:1]
Transfection of a miR-31 mimic resulted in at least 25-fold increased levels of miR-31 in the difficult to transfect HBEC line (Fig. 6e). [score:1]
Cells were then transfected with hsa-miR-31 mimic (2.5 nM) (Qiagen) or a negative control (2.5 nM) and 1.1 ul TransIT-X2 (Mirus Bio) per well in 24-well plates. [score:1]
Mo del of relationships between miR-31, cigarette smoke, and Fzd9. [score:1]
miR-31 is normalized to RNU6 and Fzd9 to GAPDH. [score:1]
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[+] score: 114
Other miRNAs from this paper: hsa-mir-29b-1, hsa-mir-200b, hsa-mir-200c, hsa-mir-200a, hsa-mir-370
Although miR-31 overexpression in a number of ovarian cancer cell lines with a dysfunctional TP53 pathway inhibited proliferation and induced apoptosis [30], its overexpression in the colon cancer cell line HT29, carrying a TP53 mutation, resulted in a strong anti-apoptotic effect. [score:8]
This suggests a possibility that the up-regulated expression of miR-200a and miR-31 during carcinogenesis plays an important role in the development of PCa by promoting the cell into the castration stage. [score:7]
The expression levels of miR-200a and miR-31 was significantly down-regulated in PCa tissues compared with matched normal glands (Fig. 3B and 3D), while there was no significant difference in the expression levels of miR-29b-1 or miR-370 (Fig. 3A and 3C). [score:7]
Additionally, up-regulated miR-31 could suppress cell cycle regulators and prevent cytotoxicity agents inducing apoptosis in PCa cells, making castration-resistant PCa therapy difficult [33, 34]. [score:7]
Chan et al. previously found that only one of three miR-31 isoforms, which differed only slightly in their 5′- and/or 3′-end sequences, directly repressed Dicer expression at both mRNA and translational levels in MCF-7 breast cancer cells and A549 lung cancer cells. [score:6]
Comparing the expression levels of miR-200a and miR-31 between primary tumor and adjacent normal glands in the present study, we observed a significant expression decrease in primary tumors, suggesting that miR-200a and miR-31 may be involved in prostate carcinogenesis. [score:5]
Lin et al. found that miR-31 expression was reduced as a result of promoter hypermethylation in primary and metastatic PCa, and that it was inversely correlated with the aggressiveness of the disease. [score:5]
However, there was no statistical association with the expression of Dicer to miR-31 expression levels (ρ [s] = -0.057, p = 0.458) (Table 1). [score:5]
Correlation studies between Dicer and miR-29b-1, miR-200a, miR-370, and miR-31 expression in PCas showed that the expression of Dicer mRNA was moderately and negatively correlated with that of miR-200a and miR-31 (ρ [s] = -0.489, p < 0.0001; ρ [s] = -0.314, p < 0.0001, respectively). [score:5]
Inhibition of miR-31 could promote in vivo metastasis through directly regulating a cohort of prometastatic genes in breast and ovarian cancers [29, 30]. [score:5]
We found that miR-31 expression level was decreased in tumor tissues compared with normal glands, indicating that miR-31 may function as a tumor suppresser gene. [score:4]
Thus, the up-regulation of miR-31 may be one reason for the failure of androgen deprivation therapy. [score:4]
Our miRNA correlation study showed that the relative expression of miR-200a and miR-31 was moderately and negatively associated with. [score:3]
Expression of miR-29b-1 (A), miR-200a (B), miR-370 (C) and miR-31 (D) in prostate adenocarcinoma and matched normal glands, after normalization to U6. [score:3]
0120159.g003 Fig 3 Expression of miR-29b-1 (A), miR-200a (B), miR-370 (C) and miR-31 (D) in prostate adenocarcinoma and matched normal glands, after normalization to U6. [score:3]
0120159.g004 Fig 4 Expression of miR-29b-1 (A), miR-200a (B), miR-370 (C) and miR-31 (D) in localized and metastatic PCa, fold changes of miRNAs levels in prostatic adenocarcinoma versus matched normal glands were log2 transformed on Y axis. [score:3]
In conclusion, our findings showed that Dicer might be a possible target for miR-200a and miR-31. [score:3]
In the present study, we examined the expression levels of miR-29b-1, miR-200a, miR-370, and miR-31 in matched PCa tissues from 185 patients. [score:3]
Taken together, both the expression and functions of miR-31 appear to be cancer-specific and possibly cell context -dependent. [score:3]
We showed that miR-200a, miR-31, and miR-370 expression levels were increased in metastatic PCa rather than in localized cancers. [score:3]
The anti-apoptotic effect of miR-31 over -expression was lower or absent in cells with a functional TP53 pathway [31]. [score:3]
Expression of miR-29b-1(A), miR-200a(B), miR-370(C) and miR-31(D) in androgen dependent and castration resistant PCa, fold changes of miRNAs levels in prostatic adenocarcinoma versus matched normal glands were log2 transformed on Y axis. [score:3]
Expression of miR-29b-1 (A), miR-200a (B), miR-370 (C) and miR-31 (D) in localized and metastatic PCa, fold changes of miRNAs levels in prostatic adenocarcinoma versus matched normal glands were log2 transformed on Y axis. [score:3]
Data analysis showed that miR-29b-1, miR-200a, miR-370, and miR-31 were down-regulated in prostate cancer samples compared with matched normal tissues. [score:3]
0120159.g005 Fig 5 Expression of miR-29b-1(A), miR-200a(B), miR-370(C) and miR-31(D) in androgen dependent and castration resistant PCa, fold changes of miRNAs levels in prostatic adenocarcinoma versus matched normal glands were log2 transformed on Y axis. [score:3]
Following the log2-transformation of miRNA fold changes in prostatic adenocarcinoma versus matched normal glands, the relative expression levels of miR-200a, miR-370, and miR-31 was higher in metastatic PCa compared with localized PCa (Fig. 4). [score:2]
Following log2 transformation of fold changes in prostatic adenocarcinoma versus matched normal glands, the relative expression levels of miR-200a and miR-31 was significantly lower in the androgen -dependent subgroup compared with the castration-resistant one (Fig. 5). [score:2]
In our study, miR-31 was elevated in metastatic PCa. [score:1]
In our cohort, the expression levels of miR-200a and miR-31 were higher in primary tumors of castration-resistant PCa compared with androgen -dependent PCa, and higher in primary tumors compared with paired normal glands. [score:1]
However, the role of miR-31 remains controversial [20– 22]. [score:1]
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[+] score: 113
Regarding colorectal cancer, we recently reported that EZH2 suppresses miR-31 expression by inducing histone H3 lysine 27 trimethylation (H3K27me3) on the miR-31 promoter and that EZH2 inhibition increased miR-31 expression [28]. [score:9]
Expression of miR-31 and efficacy of anti-EGFR therapy in KRAS (codon 12/13) wild-type group colorectal cancersIn the miR-31 low -expression group, a significantly shorter PFS (log-rank test: P = 0.022) was observed in the EZH2 low -expression group (scores of 0–1) than in the high -expression group (scores of 2–3) in Kaplan–Meier analysis (Figure 3). [score:9]
Moreover, low EZH2 expression was associated with a shorter PFS in patients with colorectal cancer, independent of gene mutations in the downstream part of the EGFR pathway and miR-31 expression, suggesting that EZH2 expression is a useful and additional prognostic biomarker for anti-EGFR therapy. [score:8]
In addition, the present multivariate analysis including the variable of EZH2 expression stratified by miR-31 and mutations of BRAF, KRAS, and NRAS showed a significantly shorter PFS in the EZH2 low -expression group than in the high -expression group. [score:8]
In the miR-31 low -expression group, a significantly shorter PFS (log-rank test: P = 0.022) was observed in the EZH2 low -expression group (scores of 0–1) than in the high -expression group (scores of 2–3) in Kaplan–Meier analysis (Figure 3). [score:7]
Figure 3Kaplan–Meier survival curves of patients treated with anti-EGFR therapeutics in the microRNA-31 (miR-31)-5p low -expression group (N = 97)(A) Progression-free survival according to EZH2 expression in the miR-31 low -expression group. [score:7]
Our current data showed a significantly shorter PFS and OS in the EZH2 low -expression group than in the high -expression group, not only in the wild-type groups of KRAS (codon 61/146), NRAS, and BRAF but also in the miR-31 low -expression group. [score:7]
Moreover, low EZH2 expression was associated with shorter PFS, independent of the mutations of BRAF (codon 600), KRAS (codon 61/146), and NRAS (codon 12/13/61) mutations and miR-31 expression. [score:7]
In the current study on patients with colorectal cancer who underwent surgical treatment, we elucidated the association of EZH2 expression, gene mutations, or miR-31 expression in the pathway downstream of EGFR with the efficacy of anti-EGFR therapy. [score:6]
Regarding the association between microRNA expression and resistance to treatment with anti-EGFR therapy, we recently suggested that high miR-31-5p expression is a useful and additional prognostic biomarker for anti-EGFR therapy [19]. [score:5]
Similarly, in the miR-31 low -expression group, a significant difference was observed in OS according to EZH2 expression (log-rank test: P = 0.048) (Figure 3). [score:5]
A high EZH2 expression was inversely associated with miR-31 expression; however, no significant relationship was found between them (P = 0.085). [score:5]
EZH2 -mediated histone methylation suppresses miR-31 expression in prostate cancer [29] and adult T-cell leukemia [26]. [score:5]
Regarding miR-31-5p expression, 12 (11%) patients and 97 (89%) patients were classified into the high- and low -expression groups, respectively. [score:5]
6.1 1.61 0.84–2.96 0.15 MicroRNA-31 expression (high -expression group vs. [score:4]
Kaplan–Meier survival curves of patients treated with anti-EGFR therapeutics in the microRNA-31 (miR-31)-5p low -expression group (N = 97). [score:3]
absent), and miR-31 (high -expression vs. [score:3]
Expression of miR-31 and efficacy of anti-EGFR therapy in KRAS (codon 12/13) wild-type group colorectal cancers. [score:3]
Unlike our previous report using colorectal cancer (N = 301) [28], no significant association was found between EZH2 and miR-31 expression, although there was a trend (P = 0.085). [score:3]
Regarding microRNA in the signaling pathway downstream of EGFR, we recently suggested that microRNA-31 (miR-31)-5p regulates BRAF activation in colorectal cancer [23, 24] and that high miR-31-5p is associated with survival in patients with colorectal cancer who underwent surgical treatment and chemotherapy with anti-EGFR antibodies [19]. [score:2]
CI, confidence interval; EGFR, epidermal growth factor receptor; EZH2, enhancer of zeste homolog 2; FFPE, formalin-fixed, paraffin-embedded; HR, hazard ratio; H3K27me3, histone H3 lysine 27 trimethylation; miR-31, microRNA-31; OS, overall survival; PFS, progression-free survival; SD, standard deviation. [score:1]
MiR-31-5p expression was analyzed by quantitative reverse transcription-PCR (qRT-PCR) using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and TaqMan microRNA Assays (Applied Biosystems) as previously described [23]. [score:1]
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[+] score: 109
Other miRNAs from this paper: hsa-mir-22, hsa-mir-101-1, hsa-mir-205, hsa-mir-101-2
As shown in Figure 6a, blocking miR-205 with an anti-miR inhibitor in WPE1-NA22 cells (a cell line that expresses high level of endogenous miR-31 [25]) decreased miR-31 expression, and increased EZH2 and E2F6 proteins. [score:7]
Subsequently, EZH2 increases trimethylated H3K27 and suppresses miR-31 expression. [score:5]
Our was performed in the revised promoter region that has no CpG island (Figure 2), indicating that EZH2 may suppress miR-31 expression independent of DNA methylation. [score:5]
EZH2 suppresses miR-31 expression. [score:5]
Similarly, depletion of EZH2 with DZNeP (a known EZH2 inhibitor [27]) increased miR-31 expression in PC-3 cells and decreased E2F6 (Figures 1c and d). [score:5]
As expected, overexpression of EZH2 suppressed miR-31 (Figure 4b), and increased E2F6 protein (Figure 4a). [score:5]
[30] We hypothesized that the epigenetic silencing of miR-205 (through promoter methylation) will lead to increased expression of EZH2, which in turn epigenetically represses miR-31 expression through histone methylation. [score:5]
As miR-31 regulates apoptosis by targeting the antiapoptotic protein E2F6, [25] we hypothesized that by silencing miR-31, EZH2 may regulate apoptosis in prostate cancer cells. [score:5]
In DU-145 cells, we also observed that siRNA knockdown of EZH2 increased miR-31 expression (Supplementary Figure 3B), docetaxel -induced apoptosis, and active caspase-3/caspase-9, as well as PARP cleavage (Supplementary Figures 1C and D). [score:4]
In another prostate cancer cell line DU-145, we also observed that siRNA knockdown of EZH2 or DZNeP treatment increased miR-31 expression and decreased E2F6 (Supplementary Figures 1A and B). [score:4]
EZH2 knockdown increased miR-31 expression with or without docetaxel treatment (Supplementary Figure 3A). [score:4]
We found that siRNA knockdown of EZH2 restored miR-31 expression in PC-3 cells (Figures 1a and b). [score:4]
We reported previously that miR-31 expression was decreased in prostate cancer cells, resulting in resistance to apoptosis. [score:3]
We have previously shown that miR-31 targets E2F6. [score:3]
In contrast, overexpression of miR-205 in PC-3 cells caused a decrease of EZH2 and an increase of miR-31, which in turn decreased E2F6 (Figure 6b). [score:3]
[26] To understand the mechanism of miR-31 silencing in prostate cancer, we examined if miR-31 is suppressed by EZH2. [score:3]
MiR-205, EZH2, and miR-31 expression in human prostate cancer specimens. [score:3]
To test our hypothesis, we examined the effects of miR-205 on miR-31 expression. [score:3]
As shown in Supplementary Figure 2, siRNA knockdown of EZH2 decreased the binding of EZH2 to the miR-31 promoter, whereas the levels of histone H3 on the promoter were not changed by EZH2 knockdown. [score:3]
We analyzed miR-205, EZH2, and miR-31 expression in eight pairs of human prostate cancer specimens and the adjacent non-malignant tissues using real-time PCR. [score:3]
Interestingly, the expression levels of miR-205, EZH2, and miR-31 correlate well among the individual patients (Pearson's correlation coefficient test: R=−0.7911 between the levels of EZH2 and miR-31; R=−0.6236 between EZH2 and miR-205). [score:3]
As a result, the antiapoptotic protein E2F6 (a target of miR-31 [25]) was decreased by EZH2 siRNA treatment. [score:3]
We found that miR-205 and miR-31 expression levels were decreased in the cancer samples compared with the normal tissues (Figure 7). [score:2]
EZH2 regulates histone methylation on the miR-31 promoter. [score:2]
In both PC-3 and DU-145 cells, miR-31 levels were not affected by E2F6 knockdown or docetaxel treatment (Supplementary Figure 5). [score:2]
MiR-205 regulates miR-31 through EZH2. [score:2]
For example, low levels of miR-205 in patients PR2647 and PR1107 match to the high levels of EZH2 in the same patients, which in turn lead to the very low levels of miR-31 in these patients. [score:1]
The transcription start site of miR-31 pri-miRNA was identified by experiments with FirstChoice RLM-RACE Kit from Ambion, using total RNA isolated from WPE1-NA22 cells as template. [score:1]
To determine if EZH2 regulates histone H3K27 methylation, we performed chromosome immunoprecipitation (ChIP) assay near the transcription start site (~360 bp upstream) on the miR-31 promoter. [score:1]
As presented in a conceptual mo del (Figure 8), we propose that EZH2 coordinates the silencing of the proapoptotic miR-205 and miR-31. [score:1]
We performed 5′ rapid amplification of cDNA ends (5′ RACE) experiments to identify the transcription start site and the promoter for the miR-31 gene. [score:1]
TM509 for miR-205, TM1100 for miR-31, Hs00544833_m1 for EZH2) from Applied Biosystems (Foster city, CA, USA). [score:1]
The gene encoding miR-31 is located on chromosome 9p21. [score:1]
This observation supports our hypothesis that EZH2 may coordinate the silencing of miR-205 and miR-31 in prostate cancer. [score:1]
12, 37 EZH2 may also contribute to apoptosis resistance by linking the genomic loss of miR-101 to miR-31 silencing. [score:1]
Although a previous study has reported promoter methylation of the miR-31 gene, [40] the methylation analysis was carried out in the region of the CpG island that is located in the intron region between exons 1 and 2 (Figure 2a). [score:1]
Thus, DNA methylation -mediated silencing of miR-205 [25] can lead to histone methylation -mediated silencing of miR-31, with EZH2 as the coordinator of the two separate events. [score:1]
The transcription start site for miR-31 was identified (Figure 2a). [score:1]
[25] It was recently shown that in adult T-cell leukemia, [26] the PRC can be recruited to the miR-31 promoter (on chromosome 9q21) by transcription factor YY1. [score:1]
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[+] score: 101
The pooled HR (hazard ratio) of OS was 1.55 (95% CI 1.30-1.86) for high versus low circulating miR-31 expression (Figure 5), so high miR-223 expression significantly decreased the OS time. [score:5]
Furthermore, high miR-31 expression significantly increased the risk of OS, although high circulating miR-31 expression was not significantly associated with poor differentiation and LNM in lung cancer. [score:5]
The eligible articles should meet the following criteria: 1) the expression of circulating miR-31 was analyzed by detection/diagnosis or prognosis of cancer in human, 2) for the prognosis analysis, patients were divided into high and low expression groups by the level of circulating miR-31, 3) diagnostic test indexes for detection/diagnosis (sensitivity, specificity, and AUC) or HRs for survival (overall survival [OS], relapse-free survival [RFS], tumor-special survival [TSS], progression-free survival [PFS]) or odd ratios (ORs) for differentiation/LNM were provided or could be calculated from the available data; and 4) the expression of circulating miR-31 was tested by RT-PCR or fluorescence in-situ hybridization. [score:5]
English or Chinese studies on the role of circulating miR-31 expression in the development of human cancer were searched in EMBASE, Cochrane Library, PubMed, Wanfang databases, and China National Knowledge Infrastructure with key words (cancer or carcinoma or tumor or neoplasm or adenocarcinoma) and (microRNA-31 or miRNA-31 or miR-31) and (serum or sera or blood or plasma and “circulating”). [score:4]
For the 5 prognosis analyses, the pooled HR (hazard ratio) of overall survival (OS) was 1.55 (95% CI 1.30-1.86) for high versus low circulating miR-31 expression. [score:3]
For the cancer prognosis, each of the 5 studies was sequentially excluded; high miR-31 expression still significantly increased the risk of OS throughout (data not shown). [score:3]
For miR-31, some meta-analyses approved that it up-expressed in cancer tissues and was associated with prognosis [8, 56]. [score:3]
As miR-31 affects multiple targets simultaneously [7], the role of miR-31 depends on the total level and differential distribution. [score:3]
However, high expression of circulating miR-31 did not significantly increase the risk of poor differentiation (pooled OR=1.39, 95% CI: 0.56-3.47) and LNM (pooled OR=3.46, 95% CI: 0.96-12.42) in lung cancer. [score:3]
Furthermore, the reference for all effects of prognosis (ORs or HRs) was reformatted as low circulating miR-31 expression, and the multivariate analysis effects were chosen for pooled analysis. [score:3]
Abnormal expression of miR-31 in tumorous tissue has confirmed it involved in tumorigenesis and progression of cancers [14– 16]. [score:3]
Forest plot of association between circulating miR-31 expression and OS. [score:3]
Figure 6 This current study aimed to assess the pooled effect of circulating miR-31 expression on detection and prognosis of cancer. [score:3]
MiR-31, a highly evolutionarily conserved miRNA, plays an important regulating role in embryonic implantation, development, bone and muscle homeostasis, and immune function [7]. [score:3]
Research in the mechanism of miR-31 found that it could target genes such as ARID1A [33], SATB2 [34], ARID1A [35], HuR [36], BAP1 [37], EZH2 [38], and RASA1 [39], and it could further activate oncogenes and promote tumor cell proliferation, migration and invasive capability in vitro. [score:3]
High expression of circulating miR-31 did not significantly increase the risk of poor differentiation (pooled OR=1.39, 95% CI: 0.56-3.47) and LNM (pooled OR=3.46, 95% CI: 0.96-12.42) in lung cancer (Supplementary Figure 3). [score:3]
The expression of circulating miR-31 might be an effective biomarker for surveillance of cancer. [score:3]
Abundant studies have reported that miR-31 was dysregulated in various human cancers, such as lung cancer [8], colorectal cancer [9], oral squamous cell carcinoma [10], cervical cancer [11], ovarian cancer [12], and upper tract urothelial carcinoma [13]. [score:2]
was conducted for the association between circulating miR-31 and cancer detection as well as cancer prognosis. [score:1]
On the other hand, the pooled HR on OS showed circulating miR-31 was also an effective biomarker for prognosis surveillance of cancer patients. [score:1]
As was no significant heterogeneity among the different cancers, it epidemiologically confirmed that circulating miR-31 might have an identical effect on prognosis of cancer patients according to the same mechanism introduced above. [score:1]
The present meta-analysis aimed to explore the effect of circulating miR-31 on cancer detection and prognosis. [score:1]
To our best knowledge, this is the first meta-analysis to confirm the significant effect of circulating miR-31 on cancer detection and prognosis. [score:1]
There were 3 and 4 studies reported the cases of poor differentiation and the cases of lymph node metastasis (LNM) by circulating miR-31 level, and all of them were the lung cancer patients. [score:1]
Circulating miR-31 was found to be associated with cancers detection and prognosis. [score:1]
Furthermore, circulating miR-31 was found associated with prognosis such as metastasis and survival [18, 19, 22]. [score:1]
Association between circulating miR-31 and OS. [score:1]
Similar to being tested in cancer tissues, miR-31 could be steadily detected in circulating blood. [score:1]
This meta-analysis is the first to demonstrate that the circulating miR-31 has relatively high effect on cancer detection and prognosis surveillance. [score:1]
Consequently, circulating miR-31 was used as a noninvasive biomarker for cancer detection and diagnosis [17– 22]. [score:1]
Circulating miR-31, always positively correlated with the level of tumor tissue [8, 47, 48], effectively represented the total level and was predicted as a good biomarker for cancer diagnosis and prognosis surveillance [7]. [score:1]
Similar to miR-21 [51], miR-223 [52], and miR-378 [53], the pooled diagnostic value of circulating miR-31 was higher than traditional clinical markers such as CEA and CA19-9. In addition, the Fagan's nomogram showed circulating miR-31 could raise the probability of cancer detection by 29% (post-test probability49% - pre-test probability 20%)[54]. [score:1]
The level of miR-31 was detected in circulating blood by RT-PCR. [score:1]
Because of too few studies of circulating miR-31 on relapse-free survival (RFS), tumor-special survival (TSS), and progression-free survival (PFS), treatment-free survival (TFS), etc. [score:1]
The general data was extracted by 3 authors (YC, HJ, ZS, HW) according to the following form: 1) basic information (first author's name, published year, region of cohort, cancer type, testing method of miR-31), 2) diagnostic test information (sample size, AUC, sensitivity, and specificity), 3) prognosis information (cases in each group of miR-31 (high/low), cases of differentiation/LNM in each group, and survival results [OS, RFS, TSS, PFS]). [score:1]
for the association between circulating miR-31 and AUC was checked by a Begg's funnel plot under the random-effects mo del. [score:1]
Circulating miR-31 is an effective biomarker and could be used as a component of miRs signature for cancer detection and prognosis surveillance. [score:1]
Meanwhile, both the pooled sensitivity and specificity being 0.79 (0.76-0.82) showed circulating miR-31 had a relatively high accuracy in human cancer detection. [score:1]
MiR-31, a common oncomiR, has been reported to increase the risk of different types of cancer. [score:1]
However, the effect of circulating miR-31 on cancer diagnosis and prognosis is controversial, and no meta-analysis has investigated the association between circulating miR-31 expression and diagnosis as well as prognosis of cancer. [score:1]
Due to circulating miR-31 acting as a diagnostic biomarker of cancer [31, 32], publication bias for test accuracy was checked by a Deek's funnel plot in the 9 tests (Figure 6) and no significant bias existed (t = 1.14, P = 0.292). [score:1]
The SROC curve of circulating miR-31 test for the diagnosis of various cancers. [score:1]
Third, in spite of the fact that the present study yielded a relatively high diagnostic value, the effect of circulating miR-31 was not high enough according to the criteria of high accuracy (PLR > 10, NLR < 0.1). [score:1]
The present meta-analysis aimed to explore the role of circulating miR-31 on cancer detection/diagnosis and further on prognosis surveillance of patients. [score:1]
For no significantly pooled effects of circulating miR-31 on differentiation and LNM in lung cancer, it was due to the limit of relatively small sample size (for differentiation, n=464; for LNM, n=511); and it also suggested that the effect of circulating miR-31 on cancers did not only depend on influence of differentiation and LNM. [score:1]
the epidemiological evidences of circulating miR-31 on chemo-radiotherapy and others were still limited and needed to be further proved. [score:1]
To search for an applicable and feasible biomarker for detection and prognosis surveillance and to provide the epidemiological evidence for mechanism studies, we focused on the association of circulating miR-31 content on cancer detection and prognosis. [score:1]
Meta-analysis of circulating miR-31 for prognosis of human cancer. [score:1]
It all suggested that circulating miR-31 was a higher effective biomarker for human cancer detection. [score:1]
The diagnostic accuracy of miR-31 for cancers was relatively high. [score:1]
pooled AUC of circulating miR-31 test for the diagnosis of various cancers. [score:1]
Meta-analysis of circulating miR-31 for human cancer detecting. [score:1]
The circulating miR-31 was an effective biomarker for cancer detection and prognosis prediction. [score:1]
Five studies showed data for OS (overall survival) by circulating miR-31 level for 612 cancer patients. [score:1]
The miR-31 level of circulating blood was positively correlated with that in cancer tissues [17]. [score:1]
Association between circulating miR-31 and differentiation, LNM. [score:1]
Furthermore, miR-31 could contribute to the epithelial-to-mesenchymal transition (EMT)[40, 41] and involved in response to chemo-radiotherapy [42– 44]. [score:1]
Publication bias for the association between circulating miR-31 and AUC was checked by a Begg's funnel plot under the random-effects mo del. [score:1]
In the present meta-analysis, the adjusted pooled-AUC of 0.79 (95% CI: 0.73-0.86) from 12 diagnosis tests and the DOR of 16.81 (95% CI: 9.67-29.25) from 9 tests indicated that the performance of circulating miR-31 to detect cancer was high feasibility [49, 50], furthermore, there was no obvious differences among cancer types. [score:1]
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[+] score: 100
That the tumor suppressor genes Fxbw7, Pdcd4, and Stk40 were downregulated at the mRNA and protein level in marked-ZD tumor group (Figure 6) and that they were predicted to interact to alter network of target proteins [65, 66, 77] (Figure 7) provide support that miR-223, miR-21, and miR-31 have an important role in ESCC and may be useful prognostic biomarkers and therapeutic targets for ESCC. [score:10]
Analysis of esophageal expression of Fbxw7, Stk40, and Pdcd4 (respective tumor suppressor targets of miR-223, miR-31, and -21) in Zn-modulated rats at tumor endpoint. [score:7]
That the three tumor suppressor targets are predicted to interact to alter network of cancer-related proteins [65, 66, 77] provide support that miR-223, miR-21, and miR-31 have an important role in ESCC and may be useful therapeutic targets in ESCC. [score:7]
We selected 8 miRNAs in ZD3T esophageal tissue (miR-223, -21, -31, -146a, -146b, -221, -194, and -106b) and two miRNAs (miR-31, -223) in ZD6T and ZD12T esophageal tissues, Figure 4B shows that the Taqman data confirmed the upregulation of all 8 selected miRNAs in ZD3T vs ZST samples, and the upregulation of miR-223 and miR-31 in ZD6T and ZD12T samples. [score:7]
Figure 7 A. The displayed esophagus-specific nine-gene network shows predicted functional relationships among the genes that are most functionally related to Stk40, Pdcd4 and Fbxw7 (tumor-suppressor targets of miR-31, mir-21 and miR-223, respectively). [score:5]
miR-223, miR-21, and miR-31 can target many important tumor suppressor genes, including FXBW7 [25, 61], STK40 [60, 62, 63], and PDCD4 [64]. [score:5]
Notably, miR-31 and miR-146a were differentially expressed in ZD6/ZD12 esophagus as they were in ZD3 esophagus, albeit at a lower expression level. [score:5]
A. The displayed esophagus-specific nine-gene network shows predicted functional relationships among the genes that are most functionally related to Stk40, Pdcd4 and Fbxw7 (tumor-suppressor targets of miR-31, mir-21 and miR-223, respectively). [score:5]
STK40 is a known negative regulator of NF-κB mediated transcription [90] and a miR-31 direct target [60, 62, 63]. [score:5]
miR-223, miR-21, and miR-31 are the top -upregulated species in the high ESCC-burden, marked-ZD esophagus. [score:4]
The signature was defined by five top up-regulated oncogenic miRNAs (miR-31, -223, -21, -146b, -146a) [15, 16, 22– 26, 29, 30, 34, 58] that were up 4.9-3.7 fold. [score:4]
In addition, prolonged ZD by itself induced an oncogenic microRNA (miRNA) signature with miR-31 as the top upregulated species [14], a feature of human ESCCs as well [15, 16]. [score:4]
Our study suggests that miR-223, miR-31 and miR-21 alone or in combination could be used as therapeutic targets for treatment of ESCC. [score:3]
Thus, moderate and mild-ZD induces alterations in miRNA expression, including miR-31 and miR-223. [score:3]
Cellular localization of miR-223, miR-31 and miR-21 expression in human ESCC tissue. [score:3]
These findings show that moderate and mild-ZD induces alterations in miRNA expression, including miR-31 and miR-146a. [score:3]
Using the nanoString platform, miRNA expression profiles distinguished the highly preneoplastic/proliferative marked-ZD esophageal phenotype with a 5-miRNA signature (miR-31, -223, -21, -146b, -146a), from the less proliferative, mild-ZD phenotype with a 3-miRNA signature (miR-146a, -31, -223). [score:3]
In addition, miR-223 and miR-31 dysregulation is common to marked-ZD and moderate/mild-ZD tumor groups (Figure 4A). [score:2]
Recently, we demonstrated by ChIP-seq analysis that in ZD esophagus, the miR-31 promoter region and NF-κB binding site were activated, unleashing miR-31 -associated STK40-NF-κB controlled inflammatory signaling to produce a preneoplastic phenotype; Zn-replenishment restores the normal regulation of this genomic region and a normal esophageal phenotype [60]. [score:2]
All 12 cases showed intense to moderate miR-31, miR-223, and miR-21 ISH signal in near serial sections of moderately to poorly differentiated ESCC tumor samples (Figure 5). [score:1]
Following deparaffinization, rehydration in graded alcohol and proteinase K treatment, tissue sections were hybridized with miR-31 probe (20 nM), miR-223 or miR-21 probe (50 nM) in hybridization buffer (Exiqon) at 50°C - 57°C for 14 h in a hybridizer (Dako, Glostrup, Denmark). [score:1]
Localization of miR-223, miR-31, and miR-21 in human esophageal squamous cell carcinoma (ESCC) tissue by in situ hybridization (ISH). [score:1]
Among which, miR-31 [15, 16, 30, 60] and miR-223 [25, 26, 34] are oncomiRs for human ESCC. [score:1]
miRCURY locked nucleic acid (LNA)™ microRNA detection probes, namely, rno-miR-21, rno-miR-31, rno-miR-223, hsa-miR-31, hsa-miR-223, negative controls (rno-miR-31) with mismatches at two position, were purchased from Exiqon (Vedbaek, Denmark). [score:1]
A Venn diagram (Figure 4A) showed that miR-31, -223, -7i, -543 were the four common miRNAs shared among ZD3T, ZD6T and ZD12T esophagus. [score:1]
Figure 4 A. Venn diagram showing miR-223 and miR-31 are common to ZD3T, ZD6T, and ZD12T esophagi (cutoff point of P < 0.05 and fold difference >1.3), and scatterplot showing their fold change vs ZST. [score:1]
A limitation of this study is the fact that the underlying biological mechanisms of the key dysregulated miRNAs in ESCC development, namely, miR-223, miR-21, and miR-31, were not investigated. [score:1]
In situ hybridizationmiRCURY locked nucleic acid (LNA)™ microRNA detection probes, namely, rno-miR-21, rno-miR-31, rno-miR-223, hsa-miR-31, hsa-miR-223, negative controls (rno-miR-31) with mismatches at two position, were purchased from Exiqon (Vedbaek, Denmark). [score:1]
A. Venn diagram showing miR-223 and miR-31 are common to ZD3T, ZD6T, and ZD12T esophagi (cutoff point of P < 0.05 and fold difference >1.3), and scatterplot showing their fold change vs ZST. [score:1]
Previously, we demonstrated an abundant miR-31 ISH signal in human ESCC tissue [60]. [score:1]
miR-31 acts as an oncomiR in squamous cell carcinomas (SCCs), including ESCC [15, 16], tongue SCC [87], head and neck SCC [88], and skin SCC [89]. [score:1]
B. Validation of eight representative miRNAs in ZD3T esophagus; and miR-223 and miR-31 in ZD6T and ZD12T esophagi. [score:1]
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[+] score: 82
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7e, hsa-mir-20a, hsa-mir-21, hsa-mir-23a, hsa-mir-24-1, hsa-mir-24-2, hsa-mir-26b, hsa-mir-27a, hsa-mir-29a, hsa-mir-29b-1, hsa-mir-29b-2, hsa-mir-103a-2, hsa-mir-103a-1, hsa-mir-199a-1, hsa-mir-148a, hsa-mir-7-1, hsa-mir-7-2, hsa-mir-7-3, hsa-mir-10b, hsa-mir-181a-2, hsa-mir-181b-1, hsa-mir-181c, hsa-mir-199a-2, hsa-mir-199b, hsa-mir-203a, hsa-mir-204, hsa-mir-212, hsa-mir-181a-1, hsa-mir-221, hsa-mir-23b, hsa-mir-27b, hsa-mir-128-1, hsa-mir-132, hsa-mir-133a-1, hsa-mir-133a-2, hsa-mir-143, hsa-mir-200c, hsa-mir-181b-2, hsa-mir-128-2, hsa-mir-200a, hsa-mir-30e, hsa-mir-148b, hsa-mir-338, hsa-mir-133b, dre-mir-7b, dre-mir-7a-1, dre-mir-7a-2, dre-mir-10b-1, dre-mir-181b-1, dre-mir-181b-2, dre-mir-199-1, dre-mir-199-2, dre-mir-199-3, dre-mir-203a, dre-mir-204-1, dre-mir-181a-1, dre-mir-221, dre-mir-222a, dre-let-7a-1, dre-let-7a-2, dre-let-7a-3, dre-let-7a-4, dre-let-7a-5, dre-let-7a-6, dre-let-7b, dre-let-7e, dre-mir-7a-3, dre-mir-10b-2, dre-mir-20a, dre-mir-21-1, dre-mir-21-2, dre-mir-23a-1, dre-mir-23a-2, dre-mir-23a-3, dre-mir-23b, dre-mir-24-4, dre-mir-24-2, dre-mir-24-3, dre-mir-24-1, dre-mir-26b, dre-mir-27a, dre-mir-27b, dre-mir-29b-1, dre-mir-29b-2, dre-mir-29a, dre-mir-30e-2, dre-mir-101b, dre-mir-103, dre-mir-128-1, dre-mir-128-2, dre-mir-132-1, dre-mir-132-2, dre-mir-133a-2, dre-mir-133a-1, dre-mir-133b, dre-mir-133c, dre-mir-143, dre-mir-148, dre-mir-181c, dre-mir-200a, dre-mir-200c, dre-mir-203b, dre-mir-204-2, dre-mir-338-1, dre-mir-338-2, dre-mir-454b, hsa-mir-181d, dre-mir-212, dre-mir-181a-2, hsa-mir-551a, hsa-mir-551b, dre-mir-31, dre-mir-722, dre-mir-724, dre-mir-725, dre-mir-735, dre-mir-740, hsa-mir-103b-1, hsa-mir-103b-2, dre-mir-2184, hsa-mir-203b, dre-mir-7146, dre-mir-181a-4, dre-mir-181a-3, dre-mir-181a-5, dre-mir-181b-3, dre-mir-181d, dre-mir-204-3, dre-mir-24b, dre-mir-7133, dre-mir-128-3, dre-mir-7132, dre-mir-338-3
Upregulation of miR-21, miR-31 and miR-181c leads to the downregulation of inhibitors and suppressors such as pdcd4, tgfbr2, bcl2l13, rgs5 and chka, downregulated genes with anti-proliferative functions. [score:14]
Fig 7 summarizes the gene regulatory circuit for miR-21, miR-31 and miR-181c, the 3 validated, shared upregulated miRNAs, with downregulated putative target genes that have functional relationships with conserved blastema -associated genes. [score:10]
0157106.g007 Fig 7Following injury, three miRNAs (miR-21, miR-31 and miR-181c) are commonly upregulated and target a set of five commonly downregulated genes shown with blue lines. [score:9]
Following injury, three miRNAs (miR-21, miR-31 and miR-181c) are commonly upregulated and target a set of five commonly downregulated genes shown with blue lines. [score:9]
S22 Table Zebrafish Ensembl gene identifiers for 58 genes downregulated in three mo dels with predicted miRNA binding sites for miR-21, miR-181c, miR-181b, miR-31 and miR-7 and members of the network of commonly up- and downregulated genes with functional interactions to 11 blastema -associated genes. [score:7]
These studies confirmed miR-21, miR-181c and miR-31 were consistently upregulated in all three organisms and miR-181b and miR-7b were upregulated in both zebrafish and bichir (Fig 3). [score:7]
These filtering criteria identified 136 downregulated genes with predicted binding sites in the 3’-UTRs for any of the 5 common upregulated miRNAs (miR-21, miR-31, miR-181b, miR-181c and miR-7b) (S21 Table). [score:7]
Morphological and histological studies of miR-21, miR-31 and/or miR-181 inhibition combined with identification of target genes would demonstrate their roles in blastema formation. [score:5]
S21 Table Zebrafish Ensembl gene identifiers for 136 genes downregulated in three mo dels with predicted miRNA binding sites for miR-21, miR-181c, miR-181b, miR-31 or miR-7 in all three mo dels. [score:4]
Within this subset of differentially regulated zebrafish miRNAs, we identified 10 miRNAs: miR-21, miR-181c, miR-181b, miR-31, miR-7b, miR-2184, miR-24, miR-133a, miR-338 and miR-204, that showed conserved expression changes with both bichir and axolotl regenerating samples (Table 1). [score:4]
STRING interactions with 11 common blastema -associated genes, miR-21, miR-31, miR-181, and 50 additional common differentially expressed genes with common predicted miRNAs binding sites. [score:3]
Next, we established a gene network for common miRNA target genes for miR-21, miR-31 and miR-181. [score:3]
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[+] score: 81
In a previously published dataset of gene expression from human keratinocytes transfected with a miR-31 inhibitor, 96 genes were up-regulated, and of these, 24 were predicted targets of miR-31 [47]. [score:10]
Of these, miR-31 is potentially the most promising based on prior work demonstrating that miR-31 is highly expressed in airway epithelia [54], and in skin epithelia regulates the expression of pro-inflammatory chemokines by targeting genes that regulate NF-kB activity [47]. [score:9]
Analysis of a dataset from human keratinocytes transfected with a miR-31 inhibitor revealed two target genes in common with miR-31 targets correlated with neutrophils, namely Oxsr1 and Nsf. [score:7]
Because miR-31 is expressed in airway epithelia and is predicted to target genes with known links to neutrophilic inflammation, we suggest that miR-31 is a potentially novel regulator of airway inflammation. [score:6]
These results indicate that miR-497, miR-351 and miR-31 may serve as mediators of neutrophilic inflammation by targeting genes that regulate neutrophil recruitment to the airways; predicted targets of each miRNA are listed in Additional file 9: Table S6. [score:6]
Based on our finding of significant enrichment of predicted miR-31 targets in our data compared to dataset from human keratinocytes in which miR-31 was inhibited [47], we suggest that two genes, Oxsr1 and Nsf, may be novel regulators of neutrophilic inflammation in the airway. [score:5]
We also found that the expression of Oxsr1 and Nsf was negatively correlated with miR-31 expression (r = −0.26, p = 2.0 × 10 [−3] and r = −0.23, p = 8.7 × 10 [−3], respectively). [score:5]
Points in red indicate miRNAs with p-values that remain significant after adjusting for multiple testing at an FDR < 0.05 miR-31 has been previously reported as a marker and/or regulator of other inflammatory conditions, such as inflammatory bowel disease [46] and psoriasis [47]. [score:4]
Given these data, one simple hypothesis is that NSF in airway epithelium regulates the release of a chemokine involved in neutrophil recruitment, and this pathway is targeted by miR-31. [score:4]
In the second approach, we constructed putative miRNA/mRNA regulatory networks and identified three miRNAs (miR-497, miR-351 and miR-31) as candidate master regulators of genes associated with neutrophil recruitment. [score:3]
Thus we conclude that miR-31 is likely to be an important modulator of neutrophilic airway inflammation in part by targeting Oxsr1 and Nsf. [score:3]
We found that the mouse orthologs of two of these genes, Oxsr1 and Nsf, were also present in our list of predicted miR-31 targets correlated with neutrophils, resulting in statistically significant enrichment (p = 4 × 10 [−3] by hyper-geometric test). [score:3]
Additionally, miR-31 is not highly expressed in neutrophils at baseline or after bacterial challenge [55], suggesting that miR-31 is not simply a surrogate metric of neutrophil counts. [score:3]
In combination with prior work in human airway and skin epithelia, our result suggests that miR-31 may be targeting genes both in the airway epithelia and in white blood cells (neutrophils, monocytes, and dendritic cells), and provide rationale for additional studies on the role of miR-31 in neutrophilic airway inflammation. [score:3]
Our results also indicate that other genes with established relationships to neutrophil recruitment are predicted target genes of miR-31. [score:3]
For the set of genes that were positively correlated with neutrophils (n = 674 at FDR < 0.1), we identified miR-497, miR-351 and miR-31 as candidate regulatory hubs (Fig.   7). [score:2]
We identified three miRNAs, miR-497, miR-351 and miR-31, that are candidate master regulators of genes associated with neutrophil recruitment. [score:2]
The bioinformatic analysis we conducted to identify miRNAs that may act as key regulators of genes involved in granulocyte (eosinophil and neutrophil) recruitment pointed to three miRNAs of interest for neutrophils, namely miR-497, miR-351 and miR-31. [score:2]
We note that many previous studies have documented an important role for miR-31 in multiple cancer processes [63]; thus our findings suggest that miR-31 is microRNA with pleiotropic effects. [score:1]
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[+] score: 79
In particular, the following target proteins, involved in cell proliferation, were downregulated: DHFR targeted by miR24 [33], Cyclin D1 targeted by miR223 [34], and E2F-2 targeted by miR31 [35]. [score:12]
The most convincing results were obtained with inhibitors of miR31 and 223 that were downregulated also in MV DCR− as well as with the inhibitor of miR451, that has been previously described as a Dicer independent miRNA [56]. [score:8]
Supporting Information Figure 3B shows that MDR1, MIF, and RAB14, targeted by miR451, and E2F-2, targeted by miR31, were overexpressed at RNA level in HepG2 with respect to hepatocytes. [score:7]
The prominent role of miR451 and miR31 transfer was indicated by the direct antitumor activity of miR451 and miR31 mimics on HepG2 that resulted in MDR1, MIF, RAB14, and E2F-2 downregulation similar to that of MV-HLSC. [score:5]
Treatment with anti-miR451 and anti-miR31 in the absence of MVs did not interfere with target expression by hepatoma tumors. [score:5]
Furthermore, HepG2 transfection with miR451 and miR31 mimics resulted in MDR1, MIF, RAB14, and E2F-2 downregulation similar to that of MV-HLSC (Fig. 5D, 5E). [score:4]
In particular, we analyzed the involvement in MV-HLSC antitumor activity of miR31 and miR451, previously described as regulators of proliferation and as tumor suppressors in different cancer cells [35, 38, 39]. [score:4]
Transfection of HepG2 with anti-miR31 (anti-miR31+MV) abrogated the E2F-2 downregulation induced by MV-HLSC. [score:4]
HepG2 transfection with miR451, miR31, and miR223 mimics, which reproduce mature endogenous miRNAs, inhibited proliferation of HepG2 (Fig. 5B). [score:3]
The inhibitory effect of MV-HLSC was abrogated by anti-miR451 and anti-miR31 administration (Fig. 6E, 6F). [score:3]
Treatment with anti-miR31 without MV administration (anti-miR31) did not interfere with E2F-2 expression by HepG2. [score:3]
Moreover, the use of miRNA inhibitors against miR451, miR223, miR24, miR125b, and miR31 on HepG2 reduced the proapoptotic activity induced by MV-HLSC. [score:3]
Similar results were observed for E2F-2, which is targeted by miR31 [38], indicating the relevance of these miRNAs in the antitumor activity of MVs. [score:3]
In mice treated with anti-miR451 and anti-miR31, the inhibition of tumor growth induced by MV-HLSC was significantly less effective than in animals treated with anti-CTR (Fig. 6, A– 6C). [score:3]
In Vivo Biological Effect of Anti-miR451 or Anti-miR31 Inhibitors. [score:3]
Similar results were observed for E2F-2 which is targeted by miR31 (Fig. 5E). [score:3]
Among miRNAs present in MV-HLSC [10], several ones were associated with potential antitumor activity, such as miR451, miR223, miR24, miR125b, miR31, miR214, and miR122. [score:1]
To evaluate whether single miRNAs with antitumor activity (miR451, miR223, miR24, miR125b, and miR31) were relevant for the proapoptotic effect of MV-HLSC, we transfected HepG2 with selected miRNA inhibitors (Fig. 5A). [score:1]
MVs released from DCR-Kd HLSC (MV DCR−), but not from CTR-A HLSC (MV CTR-A), showed a significant reduction of miR223, miR24, miR31, miR122, and miR214 as detected by qRT-PCR (Fig. 4B). [score:1]
Silencing Dicer in HLSC resulted in the modulation of different miRNAs, with a significant reduction of the antitumor miR223, miR24, miR31, and miR122 [55] in MVs. [score:1]
This was more significant for miR451, miR223, and miR31. [score:1]
Among miRNAs present in MV-HLSC, we detected several miRNAs with potential antitumor activity including miR451, miR223, miR24, miR125b miR31, and miR122 (Fig. 3A). [score:1]
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[+] score: 78
The aims of the present study were to: 1) perform a systematic investigation of the expression of ten candidate miRNAs (miR-22, miR-24, miR-31, miR-106a, miR-125b, miR-137, miR-205, miR-214, miR-221, miR-410) in human HF samples; 2) correlate these data with corresponding HF mRNA expression levels; and 3) test the identified target genes for enrichment in pathways and protein networks in order to delineate regulatory interactions in the human HF. [score:6]
Collectively, the identified target genes and pathways indicate that miR-31 is a potent cross-species inhibitor of the anagen phase. [score:5]
Expression in the human HF was confirmed for seven of the ten candidate miRNAs, and numerous target genes for miR-24, miR-31, and miR-106a were identified. [score:5]
In the miRWalk2.0 [12] and TargetScan7.0 [13] analyses, 40%, 62%, and 42% respectively of the identified target genes for miR-24, miR-31 and miR-106a were not predicted by either tool. [score:5]
Significantly correlated target genes of miR-24, miR-31, miR-106a, and miR-221. [score:3]
Intriguingly, ten of the identified target genes were shared between miR-31, miR-24, and miR-106a, suggesting that they may be critical points in the signalling cascades that control HF biology. [score:3]
The largest overlap in target genes was detected for miR-31 and miR-106a (n = 29). [score:3]
Moreover, miR-31 target genes were enriched in PDGF (Platelet-Derived Growth Factor), adipogenesis, and JAK/STAT signalling, which have been implicated previously in the control of the HF cycle [29– 33]. [score:3]
For miR-24, miR-31, and miR-106a several target genes and pathways of interest were identified (Table  1). [score:3]
The largest overlap was found between target genes of miR-31 and miR-106a (n = 29). [score:3]
Significant correlation between miRNA and mRNA expression was observed for miR-24, miR-31, miR-106a, and miR-221. [score:3]
MiR-31, miR-24 (i. e., miR-24-3p, miR-24-2-5p), and miR-106a shared the following ten target genes: FZD7, JUN, MEIS2, TAX1BP3, RBM17, SFRP1, TP63, SMARCA4, COL17A1, and ZCCHC11. [score:3]
In the PANTHER analysis, ‘Integrin Signalling’ was the top pathway for the target genes of miR-24, miR-31 and miR-106a. [score:3]
Prediction of significantly correlated target genes of miR-24, miR-31, miR-106a, and miR-221. [score:3]
Research has shown that miR-31 is responsible for both anagen inhibition and normal hair shaft formation [4]. [score:3]
For instance, miR-137 is reported to be responsible for coat colour determination in mice [5], while the inhibition of miR-31 in murine skin has been shown to result in accelerated anagen progression and abnormal hair shaft morphology [4]. [score:3]
The present analyses identified a total of 99 target genes that may act downstream of miR-31 in these processes. [score:3]
The same ten target genes were shared between miR-31 and miR-24. [score:3]
PPIs of significantly correlated target genes a miR-24; b miR-106a; and c miR-31. [score:3]
Fig. 1Overview of all target genes with a significant correlation to miR-24, miR-31, and miR-106a. [score:3]
No overlap was found for miR-221 and the three remaining miRNAs In the investigation of a potential enrichment of miRNA target genes in biological pathways, IPA revealed the strongest enrichment of the respective target genes in ‘Hepatic Fibrosis/Hepatic Stellate Cell Activation’ (miR-24), and ‘JAK/STAT Signalling’ (miR-31 and miR-106a). [score:3]
Interestingly, target genes of miR-31 were enriched in PPAR and RAR/RXRA signalling, thus supporting the hypothesis that RXRA -mediated signalling is important for the control of anagen initiation. [score:3]
However, functional studies are required to confirm the interaction between miR-31 and these pathways, and to elucidate their role in anagen control in the human HF. [score:1]
For miR-31, a total of 99 genes (53 neg. [score:1]
Ten genes (FZD7, JUN, MEIS2, TAX1BP3, RBM17, SFRP1, TP63, ZCCHC11, COL17A1, SMARCA4) were significantly correlated with miR-24, miR-31, and miR-106a (Fig.   1, Additional file 1: Table S1). [score:1]
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[+] score: 75
RQ are obtained using average of values of all miRNA for normalizer miR-708-5p, miR-31-5p, and miR-34c-5p target 3′UTR sequences of NOS1 geneTo select miRNAs that could modulate nNOS expression, the total sequence of the 3′UTR of the NOS1 gene (NOS1-3′UTR) was submitted to two predictive software, i. e., TargetScan Human and microRNA. [score:7]
The reduction in the nNOS level was confirmed by Western blot experiments showing a decrease of about 30% of nNOS expression in cells overexpressing miR-708 or miR-34c, while no significant decrease could be observed in overexpressing miR-31 (Fig.   5c). [score:7]
Surprisingly, our data revealed a slight decrease of nNOS expression by miR-31 overexpression in control human myoblasts. [score:5]
Nevertheless, no decrease of the reporter gene was observed when miR-212 was co -transfected with the parts #1, #2, #3, nor #4. These results demonstrated that miR31, miR-708, and miR34c, but not miR-212, were able to target NOS1-3′UTR sequences leading to a decrease of the reporter gene Firefly luciferase expression. [score:5]
However, no effect on nNOS expression and location could be observed when miR-31 was overexpressed compared with myoblasts transfected with the non-specific control miRNA. [score:4]
As we found a higher expression of miR-31 in severe phenotypes than in moderate phenotypes (Fig.   2c), we assume that Cacchiarelli et al. ’s patients had moderate phenotypes and therefore might not exhibit a high level of miR-31. [score:3]
miR-31, miR-708, and miR-34c effect on nNOS expression in human myoblasts. [score:3]
Graph represents average of relative quantification of miRNA normalized on SNORD44 expression of 5 (miR-31) or 3 (miR-708 and miR-34c) independent experiments. [score:3]
Furthermore, by analyzing the sequence of the NOS1-3′UTR regarding the 4 selected miRNAs, we identified 5 sequences as potential targets of miR-31, 5 for miR-708, 9 for miR-34c, and 3 for miR-212 (Additional file  3: Table S1 and Fig.   3a). [score:3]
Overall, these results were consistent with those obtained on BMDd45-55 muscle biopsies, namely a higher level of miR-31, miR-708, and miR-34c and a decrease in the expression of nNOS, thus allowing the use of these DMDd45-52 myoblasts as a suitable in vitro cellular mo del. [score:3]
A luciferase reporter study validated the targeting of NOS1-3′UTR by miR-31, miR-708, and miR-34c. [score:3]
miR-708-5p, miR-31-5p, and miR-34c-5p target 3′UTR sequences of NOS1 gene. [score:3]
Among the selected miRNAs in our study, miR-31 was already shown to be overexpressed in mdx mice and in muscular biopsies of DMD patients [14, 23, 35]. [score:3]
In the present study, we could not exclude a link between nuclear nNOS location, HDAC2 nitrosylation, and the modulation of the miR-31, miR-708, and/or miR-34c expression. [score:3]
The fact that miR-31 could target nNOS by mRNA decay was described in human atrial myocytes from patients with atrial fibrillation [36]. [score:3]
Unlike our results, Cacchiarelli and colleagues did not observe an increase of miR-31 expression in the biopsies of BMD patients. [score:3]
A higher level of expression of the 4 miRNAs was detected in BMDd45-55 compared to control muscles with a fold change of 6.6, 4.4, 10.1, and 3.3 for miR-31, miR-708, miR-34c, and miR-212, respectively, confirming the results obtained by TLDA (Fig.   2b, Additional file  2). [score:2]
Quantification by RT-qPCR confirmed a higher level of expression for miR-31, miR-708, and miR-34c in DMDd45-52 cells compared to control with a fold change of 2.2, 2.2, and 3.8, respectively (Fig.   4a). [score:2]
Among them, only the overexpression of miR-31, miR-708, and miR-34c led to a decrease of luciferase activity in an NOS1-3′UTR-luciferase assay, confirming their interaction with the NOS1-3′UTR. [score:2]
However, we could not transfect more miR-31 because of deleterious effect of transfection on human myoblasts. [score:1]
Cells were transfected with 12.5 pg of either miR -negative control (AM17111, Ambion); miR-31, miR-34c, or miR-708 (AM17100, Ambion); or antimiR-34c or antimiR-708 (AM17000, Ambion) using lipofectamine 2000 diluted in Optimem reduced medium. [score:1]
We then confirmed that DMDd45-52 cells displayed an endogenous increased of miR-31, miR-708, and miR-34c and a decreased of nNOS expression, the same characteristics observed in BMDd45-55 biopsies. [score:1]
Each 3′UTR construction (24.5 ng) was co -transfected in 293T-HEK cells with 25 pg of either miR -negative control (AM17111, Ambion) or miR-212, miR-31, miR-34c, or miR-708 (AM17100, Ambion) using lipofectamine 2000 diluted in Optimem reduced medium. [score:1]
Our data showed a significant decrease of luciferase activity when the part #2 was co -transfected with the miR-31 and the part #3 with the miR-708 and when the parts #1, or #3, or #4 were co -transfected with the miR-34c. [score:1]
One reason could be the level of miR-31. [score:1]
miR-708 n = 7, miR-31 n = 7, and miR-34c n = 8. b nNOS immunoblot in control and DMDd45-52 cells. [score:1]
We selected 4 miRNAs (i. e., miR-31, miR-708, miR-34c, and miR-212) since they were overexpressed in muscular biopsies of BMDd45-55 patients compared to healthy subjects or in muscular biopsies of patients with severe phenotypes compared to other patients. [score:1]
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[+] score: 62
Other miRNAs from this paper: hsa-mir-21
This variation in the number of detected molecule signals can be explained by the heterogeneous expression of miR-31, as would be expected of the heterogeneous diversity of individual cancer cells. [score:3]
In the case of miR-31, for example, reports on its validation in metastasis have in part been contradictory with some studies showing an association of high miR-31 expression with poor prognosis in some cancer types [3, 34] but with a favourable prognosis in others [5, 35]. [score:3]
In one study, a low miR-31 expression was found to be essential for the transformation of normal into cancer -associated fibroblasts [36], capable of significantly enhancing progression of cancer cells towards an invasive phenotype, emphasizing that dissecting miR cross talk between different cell types at the subcellular level might be crucial in pining down their exact function in processes such as metastasis. [score:3]
Moreover, since we observed that cluster density is different in cells with different metastatic propensities, we think that the differential clustering of miR-31 single molecules in SW480 and SW620 is biologically relevant, and could be closely related, e. g., to a differential multi- target miRNA interaction with several different mRNAs. [score:3]
Additionally, as further proof of probe specificity, we conducted luciferase reporter assays in SW480 and SW620 cells using the 3′UTR of cMET mRNA, which is a validated target of miR-31 [22]. [score:2]
Some miRs, such as miR-31, are especially interesting as they are perceived to be potential master regulators of metastasis, especially in solid tumors like breast cancer [2]. [score:2]
Using SMLM, we superimposed exosome-tagged GFP signals acquired with wide-field microscopy with the reconstruction images of miR-31-molecules, enabling us to obtain an overlap of miR-31-signals within exosomes within and outside the cell. [score:1]
We also present our analysis of the subcellular distribution of detected miR-31-molecules, their clustering patterns and the co-localization of secreted molecules with exosomes, and for the first time show significant differences in the distribution of miR-31 molecules in human cancer cells with high and low metastatic potential. [score:1]
SMLM images of single cells reveal clustering of miR-31 molecules and differential extracellular distribution in low- and highly metastatic CRC cells. [score:1]
The human CRC SW480 (low metastatic potential) and SW620 (highly metastatic) cell lines were transfected with 10 nM of miR-31 probe-Alexa568 (red color) for 24 h. Then, the plasma membranes of cells were stained with Cell Mask Deep Red (purple color). [score:1]
Confocal time-lapse live-cell imaging of exosome traced CD81-GFP-SW480 and CD81-GFP-SW620 cells transfected with an Alexa568-tagged miR-31 probe. [score:1]
Taken together, our novel SMLM-approach is the first to significantly detect miR-31 at the single-cell, single-molecule level and could therefore, in contrast to conventional microscopy, be exploited to not only acquire detailed information about the subcellular localization of miR-31 or indeed any other (small) non-coding RNA in individual human cells, but to also understand their interactions in cell-cell communication at a detailed single-molecule level. [score:1]
F. Representative example of the subcellular distribution of miR-31 molecules in a cell; cytoplasm (blue), plasma membrane (red), and the SMLM reconstruction of miR-31 molecules are represented in green. [score:1]
This enhanced luciferase activity together with our RT-PCR experiments, confirms that our probe with Alexa568 is specific to miR-31, and at least at the c-Met mRNA, exerts a similar function as the endogenous miR-31 molecule (Supplementary Figure 2B). [score:1]
E. Cluster density distribution of miR-31 molecules in SW480 and SW620 cells showing a higher number of clusters/μm [2] cell surface area in SW620 cells. [score:1]
In order to acquire images, including positions of the individual miRs in fixed cells, photo-switchable visualization of the labelled miR-31 molecules was implemented. [score:1]
SW620 cells were found to have a higher number of extra-cellular miRs in both sub-populations H. Relative quantities of extracellular miR-31 molecules in the round and fibroblast shaped sub-populations of SW480 and SW620 cells. [score:1]
C. Localization accuracy histogram of miR-31 SMLM acquired molecule signals in exosomes. [score:1]
Exosome -associated localization of miR-31 in highly metastatic versus low-metastatic human colorectal cancer cells. [score:1]
Distribution of miR-31 molecules in SW480 and SW620 CRC cells by conventional microscopy, including 3D-reconstruction of confocal images. [score:1]
As was evident from the segmentation analysis, both SW480 and SW620 cell lines had relatively large numbers of detected miR-31 signals in the extracellular space. [score:1]
These data show that whereas both SW480 and SW620 cells generate exosomal microRNAs, the SW480 cells produce exosomes with a higher variation per fraction of miR-31 molecules. [score:1]
Similar results were obtained for miR-21 with the exception that both cell lines had lower cluster density than was observed for miR-31(Supplementary Figure S5). [score:1]
To define potential miR-31 clusters, we used home-written software in Matlab, as described by Kaufmann et al. [23]. [score:1]
Here, we report the first single-molecule super-resolution localization microscopy approach that is able to detect single microRNA molecules with a localization accuracy of 10–15 nm, using the metastasis relevant hsa-miR-31 as a first prototype molecule. [score:1]
The different cell lines were characterized by varying levels of miR-31 endogenous expression (Supplementary Figure S2A). [score:1]
Single-Molecule Localization Microscopy and detection of miR-31 molecules in cancer cell lines. [score:1]
In comparison with random data, both cell lines showed a distribution of miR-31-molecules that was significantly non-random (Supplementary Figure S4). [score:1]
First, by using RT-PCR, we quantified the levels of miR-31 in transfected and untransfected cells. [score:1]
Both cell lines were seeded overnight and transfected with 10 nM of miR-31 Alexa568-probe in FCS -depleted media for 48 h. The exosomes were then isolated by ultracentrifugation and fixed with 4% PFA. [score:1]
To visualize and detect the selected proof-of-principle miR of interest, we transfected SW480 and SW620 cells with a linear RNA oligonucleotide probe, whose sequence was complementary to that of the human mature miR-31. [score:1]
The highly metastatic SW620 cell line had significantly more miR-31 molecules in exosomes that were predominantly seen in fractions 4 and 7, whereas in comparison, the SW480 cell line had fewer miR-31 molecules in exosomes seen across fractions 3–5. [score:1]
B. Budding of vesicle-like structures containing miR-31 molecules in CD81-GFP-SW480 and –SW620 cells during cell division. [score:1]
The most abundant fraction of miR-31 in GFP-CD81-SW480-exosomes was found in fraction 4 and that for GFP-CD81-SW620 cells was in fractions 3–5. [score:1]
In contrast, the highly metastatic SW620 cells showed a much higher total number of single miR-31 molecule signals than the SW480 cells, with a significant number in the very high range of 2.5 × 10 [4]– 4 × 10 [4] molecules/cell (Figures 2E and 2F). [score:1]
E. and F. Absolute tally (numbers) of single miR-31 molecule signals detected in SW480 and SW620 cell lines. [score:1]
Ideally, our miR-31-Alexa568 probe binds to endogenous miR-31 molecules in the cell, and consequently, less miR-31 is available to repress the reporter luciferase gene, meaning that in comparison to a scrambled oligonucleotide control, the reporter activity is increased. [score:1]
SMLM images of isolated exosomes and their co-localization with miR-31 in CD81-GFP stable -SW480 and -SW620 cells. [score:1]
The more metastatic SW620 cells, however, on average showed a higher number of miR-31 molecules across the majority of exosomal fractions. [score:1]
The SW480 low-metastatic and the SW620 highly metastatic human CRC cell lines were transfected with 10 nM of an Alexa568 labelled miR-31 probe for 24 h, and fixed with 4% PFA. [score:1]
We also observed significant heterogeneity in the number of detected single miR-31 molecule signals. [score:1]
Also, this would certainly enable the metastatic cell, rather than the non-metastatic cell, to efficiently perform long distance communication with distant cells, compartments or organs via accumulated miRNAs like miR-31 in their secreted exosomes. [score:1]
10–50 nM of labelled miR-31 probe or mimic control plasmids were co -transfected with 50 ng of the psi-CHECK-2 MET 3′UTR luciferase reporter plasmid. [score:1]
Detection and co-localization of miR-31 in isolated exosomes of highly metastatic versus low-metastatic human colorectal cancer cells. [score:1]
B. Frequency histogram of miR-31 molecules in individual clusters in the two cell line types showing a wide range in miR-molecule count. [score:1]
C. Frequency histogram of densities of miR-31 molecules within clusters in both SW480 and SW620 cells which are almost identical. [score:1]
Within individual clusters, the number of detected miR-31 single molecule signals was almost the same in both cell lines (Figure 3B), resulting in comparable miR-31 signal densities within the clusters (Figure 3C). [score:1]
The cells were transfected using Metafectene (Biontex Laboratories GmbH, Martinsried, Germany) with an SMLM suitable 5′-end photoswitchable Alexa568 fluorophore labelled RNA oligonucleotide probe, with a sequence complementary to the human mature miR-31 (IBA Gmbh, Göttingen, Germany). [score:1]
Cluster and segmentation analysis of subcellular localization of miR-31 in metastatic versus non-metastatic human colorectal cancer cells. [score:1]
The SW620 cells had on average more miR-31 molecules in the extracellular compartment than the SW480 cells (Figure 3H). [score:1]
D. Bar chart showing the number of detected miR-31 single molecule signals in the different fractions of the isolated exosomes. [score:1]
SMLM analysis of the detected miR-31 single molecule signals showed variations in the quantity of miRNA molecules within the respective fractions in both cell lines (Figure 5D). [score:1]
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Furthermore, miR-31 was not predicted to target any of the down-regulated genes, but the possibility of it affecting target genes post-transcriptionally without reducing the mRNA levels cannot be excluded. [score:8]
Two of these miRs corresponding to the same stem-loop precursor, miR-31-5p and miR-31-3p, were up-regulated by more than 10 fold while the other three (miR-338-3p, -136-5p and -10b-5p) were found to be down-regulated in DFS (Fig 1B). [score:7]
In psoriasis, miR-31 targets serine/threonine kinase 40 (STK40), a negative regulator of NFkB, which in turn results in increased expression of proinflammatory cytokines and chemokines [34]. [score:6]
miR-31-3p, miR-31-5, and miR-29c-3p expression shows up-regulation in full thickness skin samples. [score:6]
Specifically, miR-31 was recently found to be up-regulated in psoriasis, which shares some phenotypic similarities with DFUs such as a hyperproliferative epidermis, parakeratosis, and unresolved inflammation. [score:4]
0137133.g003 Fig 3 A. Both miR-31-3p and miR-31-5p show up-regulation in DFS compared to NFS, similar to what was observed in the PCR arrays generated from epidermis. [score:3]
A. Both miR-31-3p and miR-31-5p show up-regulation in DFS compared to NFS, similar to what was observed in the PCR arrays generated from epidermis. [score:3]
We found a high correlation of miR-31-3p and miR-31-5p expression between the laser captured epidermis and full thickness biopsies (n = 6, miR-31-3p Spearman’s ρ = 0.94, p = 0.017; miR-31-5p Spearman’s ρ = 0.82, p = 0.072). [score:3]
Further, when the top two up-regulated miR-31-5p and miR-31-3p were assayed in a larger sample set they did not reach statistical significance due to variable levels from patient to patient. [score:3]
Even though there is a trend towards increased expression of both miR-31-3p and miR-31-5p in DFS in comparison to NFS, this difference did not reach statistical significance (p>0.05). [score:3]
We have shown that miR-31 sustains an activated keratinocyte phenotype [35] and has been found to regulate cell differentiation and proliferation, all of which are de-regulated in DFUs [36, 37]. [score:3]
Yet, despite showing a trend of induction of miR-31 in DFS we did not observe either morphological changes in terms of differentiation or inflammation in the epidermis, or increased expression of the proliferation marker Ki67. [score:3]
However, they did not reach statistical significance when tested in a larger set of full thickness biopsies (miR-31-3p p = 0.07, miR-31-5p p = 0.31). [score:1]
Interestingly, most of the individuals with DM with the highest miR-31 levels also had renal insufficiency suggesting either longer lasting or more severe DM. [score:1]
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Meanwhile, overexpressing miR-31 reduced osterix, osteocalcin, and osteopontin protein expression without affecting RUNX2 protein levels, suggesting that miR-31 specifically influences downstream targets of RUNX2 and strongly support a role for miR-31 in osteogenesis [18, 19]. [score:7]
Such delivery systems hold promise for conditions such as age-derived osteoporosis; endothelial-secreted microvesicular miR-31, which increases with age, has been reported to inhibit osteogenesis, thus inhibition of miR-31 may reverse this effect [36]. [score:5]
Whilst the key results in this paper demonstrate a clear link between miR-31 suppression and accelerated osteogenesis, the study also revealed that efficacy varied depending on the antagomiR sequence directionality (i. e. 5A denotes the 5’ end of the antagonist sequence of miR-31, and 3A denotes the 3’ end). [score:4]
Due to the reported progressive loss of miR-31 in differentiating MSCs, we hypothesise that blocking miR-31 with antagomiRs at an early culture time point should allow an increase in osterix expression, encouraging osteogenesis. [score:3]
Herein, we identify a potential role for miR-31 in induction of osteogenesis using GNPs as a delivery platform for antagomiRs targeted against MiR-31. [score:3]
A decrease in miR-31 has recently been reported during osteogenesis, linked to osterix expression [9, 12– 17, 24]. [score:3]
The predicted structure for the sequences is shown in Fig 8, whilst the binding potential results are shown Fig 9. The binding energies of both antagomiRs for their corresponding miR-31 (miR-31-5’ with 5A and miR-31-3’ with 3A) target sequence indicated strong bond formation that required ~ -40 kcal/mol to dissociate. [score:3]
In this manuscript we have shown that by using GNPs as delivery platforms for antagomiR sequences against miR-31, we can indeed increase osterix expression in MSCs, encouraging osteogenesis. [score:3]
In the same year another group, using lipofectamine transfected cells, demonstrated that miR-31 expression was progressively decreased in human bone marrow derived stem cells undergoing osteogenesis, highlighting a potential role in differentiation [15, 16]. [score:3]
We therefore designed thiolated antagomiRs against miR-31, with both 5’ and 3’ reading direction (5A and 3A respectively), which were conjugated onto GNPs for delivery into cells. [score:2]
Aside from the principal target osterix, these additional primers were selected based on their possible link to miR-31 through osteoblast-like pathways including RUNX2, the BMPs and SMADs (intracellular signalling proteins). [score:2]
MiR-31 is hypothesised to act via suppression of a later-stage osteogenic transcription factor, osterix (downstream of runx2) [9, 12– 17]. [score:2]
The antagomiR 3’ sequence (i. e. 3A) was predicted to form a bond with the opposing 5’ sequence of miR-31 (-22.9 kcal/mol), whilst the 5’ antagomiR sequence (5A) was predicted to form a weaker structure with the opposing 3’ miR-31 sequence (-12.6kcal/mol). [score:1]
Adapted from Lain and Stein et al, and Baglìo and DeVescovi et al. When comparing miR signatures in Table 1, it is noted that miR-31 has been implicated in osteogenesis in both MSCs and pre-osteoblastic cells. [score:1]
The same principle relates to antagomiR-31 3’, which binds with perfect complementarity to the miR-31 3’ sequence. [score:1]
AntagomiR-31 5’, is designed to bind with the corresponding miR-31 5’ sequence. [score:1]
Although both the 5A and 3A antagomiRs are antagonists of miR-31, the two species are from different sections of the miR-31 sequence. [score:1]
The human osteosarcoma cell line MG63 (ATCC [®] CRL-1427 [™]) was employed in this study because of its innately high levels of miR-31. [score:1]
As expected, the nonsense strand’s ability to bind to miR-31 was even weaker. [score:1]
This differential between binding energies of the 5A and 3A with the different miR-31 stands could be a reason for the range of responses observed. [score:1]
In this regard, several recent papers demonstrated a progressive loss of miR-31 during osteogenesis; we therefore hypothesized that blocking miR-31 with antagomiRs at an early culture time point should permit an increase in MSCs committing to an osteoblastic lineage. [score:1]
Please note that 5A denotes the 5’ end of the antagonist sequence of miR-31, and 3A denotes the 3’ end of the antagonist sequence of miR-31. [score:1]
A recent report demonstrated changes in osterix when an osteosarcoma cell line, MG63 cells, (pre-osteoblastic phenotype) was treated with cholesterol -modified miR-31 sequences [12]. [score:1]
The different efficiency of 5A and 3A could also be due to differences in the structure of the primary RNA sequences, which, based on the minimum free energy method, are shown in Fig 8. MiR-31 5’ and the corresponding 5’ antagomiR (5A), when single stranded, form structures with large open unbound sequences of ~9 bp in length. [score:1]
53 Please note that 5A denotes the 5’ end of the antagonist sequence of miR-31, and 3A denotes the 3’ end of the antagonist sequence of miR-31. [score:1]
A report by Chan et al in 2013, found a range of concordant and disconcordant responses to miR-31 by shifting the sequence by one nucleotide [35]. [score:1]
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Table 1 Regulatory miRNAs involved in LCSCs MicroRNAs Source Tumor type Correlated factors Target genes Cell surface markers Signaling pathway miR-145 Human lung cancer tissues Adenocarcinoma Sox2, fascin Oct4 SP/CD133 Oct4/sox2/fascinChiou et al. (2012), Zhang et al. (2011), Hu et al. (2014), miR-31 A549cell lines/side population cells Adenocarcinoma G0/G1 phase G0/G1 phase SP/CD133/326 UnknownHua et al. (2012), Lin et al. (2012) miR-7 A549cell lines/side population cells Adenocarcinoma G1/S phase G0/G1 phase SP/CD133/326 UnknownHua et al. (2012), Lin et al. (2012) miR-34a A549cell lines/H460/H1299/mice Non-small cell lung cancer p53/BCL2 p53/BCL2 CD44/CD133 p53/Notch1/Nontch2Shi et al. (2014), Bommer et al. (2007), Li et al. (2009), Pang et al. (2010b), Balca-Silva et al. (2012) miR-200b SPC-A1/H1299/human lung cancer tissues Adenocarcinoma HDAC1/Oct-4/SOX-2/Bmi-1 Suppressor of zeste-12 (Suz-12) CD133/326 The HDAC1/miR-200b/Suz-12-E-cadherin signalingChen et al. (2014) Fig.  1 The different miRNAs regulate properties of lung cancer stem cells (LCSCs). [score:7]
miR-145 targets sites of the 3′UTRs of Oct4, Sox2, and Fascin1 and represses Oct4, Sox2, and Fascin1 to inhibit the properties of LCSCs Encoded by a single genomic locus, microRNA-31 is expressed in a variety of tissues and cell types (Grimson et al. 2007; Landgraf et al. 2007). [score:7]
Interestingly, Hua et al. (2012) found that low expression of let-7 was the key to the preservation of their ability to proliferate, and low expression of microRNA-31 was necessary for their undifferentiated status to persist. [score:5]
It has been confirmed that microRNA-31 and let-7 are significantly down-regulated in lung SP cells (lung CSCs). [score:4]
As regulators for maintaining the balance between differentiation and quiescence for SP cells, let-7 and microRNA-31 would be novel endogenous lung CSC targets to treat lung cancer. [score:4]
Similarly, down-regulation of miR-31 was observed in human bladder carcinomas with an invasive phenotype (Wszolek et al. 2011). [score:4]
MicroRNA-31 acts as an oncogenic gene, while let-7 functions as a lung cancer suppressor microRNA (Johnson et al. 2007; Liu et al. 2010), which suggests that miR-31 and let-7 have opposite functions on lung CSCs. [score:3]
Additionally, let-7 and microRNA-31 are critical for SP cells to preserve their stemness, as it was revealed that reduced miR-31 could inhibit cell proliferation by a cell cycle arrest in the G0/G1 phase, whereas reduced let-7 induced cell proliferation by accelerating the G1/S phase transition. [score:3]
Altered expression of miR-31 has been verified in various human tumours (Valastyan and Weinberg 2010). [score:3]
Antisense oligonucleotide transfection experiments have supported this view by cell cycle research that showed that reduced miR-31 could inhibit cell proliferation by a cell cycle arrest in the G0/G1 phase, whereas reduced let-7 induced cell proliferation by accelerating the G1/S phase transition (Hua et al. 2012; Fig.   3). [score:3]
In two other studies on lung adenocarcinoma CSC, let-7and miR-31 were significantly down-regulated in side population (SP) cells, which are an enriched source of CSCs, compared to non-SP cells (Hua et al. 2012). [score:3]
Fig.  3As is shown in a, let-7 and miR-31 are regulators for maintaining the balance between differentiation and quiescence for LCSCs. [score:2]
MicroRNA-31 and let-7 regulate lung CSCs. [score:2]
Valastyan et al. (2009a) has stated that miR-31 impairs more than three distinct steps of the invasion–metastasis cascade: local invasion, one or more early post-intravasation events (intraluminal viability, extravasation, and/or initial survival in the parenchyma of distant tissues), and metastatic colonization (the outgrowth of micrometastases into macroscopic secondary lesions). [score:1]
It is reported that miR-31 levels are inversely associated with the propensity to suffer metastatic relapse in primary human breast tumours (Valastyan et al. 2009b). [score:1]
Cell cycle studies further revealed that increasing miR-31 could enhance the proliferation of LCSCs by speeding up the cell cycle in the G0/G1 phase, whereas reduced let-7 induced cell proliferation of LCSCs by accelerating the G1/Sphase transition (b) The miRNA-34 family, which is composed of miR-34a, miR-34b, and miR-34c, is confirmed to be involved in the p53 and Notch signalling pathways (Shi et al. 2014; Bommer et al. 2007; Li et al. 2009; Pang et al. 2010b; Balca-Silva et al. 2012; Fig.   4). [score:1]
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Before the onset of lupus, male and female NZB/W [F1] mice have comparable levels of lupus -associated miRNAsIn our previous study where we utilized only female NZB/W [F1] mice, we reported that a set of miRNAs including the miR-96-182-183 cluster, miR-155, miR-31, miR-127, and miR-379 were upregulated only in the splenocytes from diseased 36–40-wk-old (8–9 months) female NZB/W [F1] mice, but not in the splenocytes from the pre-diseased 16–18-wk-old (3–4 months) NZB/W [F1] mice when compared to those in the control NZW mice [34]. [score:7]
In our previous study where we utilized only female NZB/W [F1] mice, we reported that a set of miRNAs including the miR-96-182-183 cluster, miR-155, miR-31, miR-127, and miR-379 were upregulated only in the splenocytes from diseased 36–40-wk-old (8–9 months) female NZB/W [F1] mice, but not in the splenocytes from the pre-diseased 16–18-wk-old (3–4 months) NZB/W [F1] mice when compared to those in the control NZW mice [34]. [score:7]
We observed an increase of miR-31 in splenocytes from murine lupus, which may contribute to autoimmunity by suppressing regulatory T cell (Treg) development or function as it was reported to target Foxp3, a lineage specific transcription factor for Tregs [47]. [score:7]
We initially analyzed the expression of lupus -associated miRNAs including the miR-96-182-183 cluster, miR-155, miR-31, miR-127, miR-379, and miR-148a in splenocytes from male and female NZB/W [F1] mice at 17–18 wks old, an age before the onset of disease in NZB/W [F1] mice. [score:5]
Our finding of increased miR-182 cluster, miR-155, miR-31, and miR-148a expression in female NZB/W [F1] mice at an age after the onset of lupus validates our previous report of the association of these miRNAs with lupus manifestation in this mo del. [score:3]
We recently reported that female NZB/W [F1] mice had increased expression of lupus -associated miRNAs such as the miR-182-96-183 cluster, miR-31, miR-155, miR-127, and miR-379 only at an age when lupus is manifested [34]. [score:3]
As shown in Figure  1A, there was no significant difference in the expression of miR-182-96-183 cluster, miR-155, miR-31, and miR-148a between male and female mice. [score:3]
However, these two estrogen-lymphoma mice displayed large variations in expression of other miRNAs such as miR-127, miR-327, and miR-31 (Additional file 1: Figure S2). [score:3]
Unlike our finding in murine lupus mo dels, human lupus peripheral blood T cells had decreased miR-31 expression, which correlated with reduced IL2 production in human lupus T cells [35]. [score:3]
Impressively, we found that after the onset of lupus, the expressions of lupus -associated miRNAs (miR-182-96-183, miR-31, miR-127, miR-379, and miR-148a, miR-155) were significantly increased in female NZB/W [F1] mice when compared to those in age-matched male mice. [score:2]
The sex differences in the expression of lupus -associated miRNAs, including the miR-182-96-183 cluster, miR-155, miR-31, miR-148a, miR-127, and miR-379, were markedly evident after the onset of lupus, especially at 30 wks of age when female NZB/W [F1] mice manifested moderate to severe lupus when compared to their male counterparts. [score:2]
There was a trend (albeit not significant) of increase of miR-31 and miR-127 expressions in 32-wk-old estrogen -treated mice when compared to 32-wk-old placebo -treated control mice. [score:2]
At 23 wks of age, the expression levels of miR-182, miR-183, miR-127, and miR-31 were significantly increased in female NZB/W [F1] mice when compared to age-matched male NZB/W [F1] mice (Figure  2A). [score:2]
Among the above lupus -associated miRNAs, miR-31 and miR-148a were reported to be dysregulated in human lupus patients and contributed to human lupus pathogenesis by affecting IL-2 production and by causing CD4 [+] T cell hypomethylation and induction of autoimmune -associated genes, respectively [35, 36]. [score:2]
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In summary, miR-200c targeted PLCγ1, PKCβII, IKKβ, and MYD88; miR-203 targeted SYK, PKCβII, MYD88, PI3K class I (α/β/δ/γ), and RasGRP3; and miR-31 targeted CD79B, PKCβII, MYD88, and NIK in B cells. [score:7]
This experimental evaluation was confirmed in T cell activation with miR-31, which has been identified as a NIK suppressor 8. Lentivirus -mediated miR-31 or shNIK expression suppressed CD25 expression and cell proliferation induced by T cell receptor stimulation in resting CD4 + T cells (Supplementary Fig. 1b,c). [score:7]
First, we co-expressed human Argonaute family proteins and miR-31 in breast cancer cell line MDA-MB-231 stably expressing Luciferase mRNA with 2 × miR-31 binding sites in 3′UTR (Luc-di-miR-31), which was used for the control experiments because of absence of endogenous miR-31 (reference 23). [score:5]
For screening of target genes of the variable miRNAs (miR [3]: miR-200c, miR-203, and miR-31), we expressed each miRNA in SUDHL8 cells and then quantified mRNA incorporated in the miRNA-abundant RISC (Fig. 6a). [score:5]
NIK mRNA was detected in Ago2 complex from normal B and T lymphocytes, but not from ATL cell line TL-Om1 that showed NIK overexpression and miR-31 loss 8 (Fig. 2i). [score:3]
Luciferase activity (n = 3) means miR-31 -dependent suppressive effect. [score:3]
Secondary structure analysis by mfold program confirmed stem-loop structure of the pre-miR-31 mutant series expressed by lentiviral vectors. [score:3]
The focal cluster (low expression in lymphoma) included miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429), miR-203, miR-205, miR-135b, and miR-31. [score:3]
Immunoprecipitation of Ago1–4 was performed by anti-FLAG antibody (a) and then co-precipitated miR-31 (b), luciferase mRNA carrying 2 × miR-31 binding sites (Luc-di-miR-31), endogenous NIK mRNA, and RPL19 mRNA (as a negative control) (c) were quantified by qRT-PCR (n = 3). [score:1]
Functional loss of miR-200c, miR-203, and miR-31 in DLBCL cells. [score:1]
RNAhybrid mo del represents hybridization between the designed miR-31 series and its corresponding perfect match sequence (d). [score:1]
Left scatter plot (g) represents the relationship among luciferase activity, miR-31 level, and luciferase mRNA level. [score:1]
The influence on miRNA function was calculated by luciferase reporter with miR-200c, miR-203, and miR-31 target sequences in 3′UTR (n = 3). [score:1]
We performed immunoprecipitation of Ago proteins by anti-FLAG antibody and then quantified co-precipitated miR-31, Luc-di-miR-31, and endogenous NIK and RPL19 (as a negative control) mRNAs. [score:1]
Symbol colors represent miR-31 condition. [score:1]
Right graph (h) shows relationship between total and RISC-incorporated miR-31 level. [score:1]
The results showed that miR-200c, miR-203, and miR-31 prevented B cell activation (Fig. 5c). [score:1]
In particular, miR-200c, miR-203, and miR-31 commonly showed significant effects (Fig. 5b). [score:1]
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The qRT-PCR analysis indicated that 2 miRNAs (miR-21 and miR-133b) were deregulated in both EAC and GC, and 6 miRNAs (up-regulated: miR-194, miR-31, miR-192, and miR-200a; down-regulated: miR-203 and miR-205) in EAC, as compared to BE but not in GC, indicating their potential unique role in EAC. [score:7]
We found that 2 miRNAs (miR-192, and miR-194) were up-regulated and 3 miRNAs (miR-205, miR-203, and miR-31) were down-regulated in BE as compared to NS (Figures 2 and 3). [score:6]
Our validation confirmed the up-regulation of miR-194, miR-192, miR-21, and miR-200a, and the down-regulation of miR-203, miR-205, miR-133b, and miR-31 in EAC as compared to NS. [score:6]
We showed the up-regulation of miR-194, miR-192, miR-21, miR-31, and miR-200a (Figure 2, Table 3) and the down-regulation of miR-133b, miR-203, and miR-205 in EAC as compared to BE (Figure 3, Table 3). [score:6]
Four miRNAs (miR-194, miR-192, miR-31, and miR-200a) were up-regulated in EAC but not in GC (Table 4, Figure 5). [score:4]
Our data showed that miR-194, miR-192, miR-21, and miR-31 were up-regulated in BE adjacent to HGD lesions relative to isolated BE samples. [score:4]
We found that miR-192, miR-194, miR-31, and miR-21 were significantly up-regulated in BE tissues adjacent to HGD, relative to the isolated BE samples (P<0.05) (Figure 8, Table 5). [score:4]
The log10 values of fold expression of the 8 miRNAs (miR-192, miR-200a, miR-194, miR-21, miR-203, miR-205, miR-31, and miR-133b) were used for hierarchical clustering. [score:3]
In addition, our data showed that miR-192, miR-194, miR-21, and miR-31 were significantly dysregulated in BE adjacent to HGD relative to isolated BE tissue samples, showing levels similar to those observed in HGD and EAC. [score:2]
0064463.g005 Figure 5The expression levels of the 6 miRNAs (miR-194, miR-192, miR-203, miR-205, miR-200a, and miR-31) were measured by means of qRT-PCR in 13 BE, 34 EAC, 45 NG, and 33 GC tissue samples. [score:1]
0064463.g003 Figure 3 The expression of 4 miRNAs (miR-205, miR-133b, miR-203, and miR-31) was evaluated using qRT-PCR in 46 NS, 13 BE, 17 HGD, and 34 EAC tissues. [score:1]
The expression of 4 miRNAs (miR-205, miR-133b, miR-203, and miR-31) was evaluated using qRT-PCR in 46 NS, 13 BE, 17 HGD, and 34 EAC tissues. [score:1]
miR-194, miR-192, miR-200a, miR-21, miR-203, miR-205, miR-133b, and miR-31 were selected for validation using 46 normal squamous (NS), 23 Barrett’s esophagus (BE), 17 Barrett’s high grade dysplasia (HGD), 34 EAC, 33 gastric adenocarcinoma (GC), and 45 normal gastric (NG) tissues. [score:1]
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[+] score: 43
Increased expression of miR-31 reduced the BAP1 expression, leading to cell proliferation and suppressed apoptosis [93]. [score:7]
Xu et al. reported that the downregulation of miR-31 enhanced lung cancer proliferation and migration by upregulating HuR, an RNA binding protein [95]. [score:7]
Edmonds et al. reported that miR-31 directly downregulated six negative regulators of the RAS/MAPK signaling pathway (SPRED1, SPRED2, SPRY1, SPRY3, SPRY4, and RASA1) and promoted mutant KRAS -mediated oncogenesis [94]. [score:6]
MiR-31 targets BAP1, which is a necessary nuclear-located deubiquitinating enzyme that acts as a tumor suppressor in lung cancer. [score:4]
Oku dela et al. reported that restoration and knockdown of miR-31 in lung cancer cell lines attenuated their growth activities and enhanced oncogenic phenotypes, respectively, suggesting that miR-31 acts as a tumor suppressor [95, 96]. [score:4]
Xu H. Ma J. Zheng J. Wu J. Qu C. Sun F. Xu S. MiR-31 Functions as a Tumor Suppressor in Lung Adenocarcinoma Mainly by Targeting HuRClin. [score:4]
MiR-31 MiR-31 targets BAP1, which is a necessary nuclear-located deubiquitinating enzyme that acts as a tumor suppressor in lung cancer. [score:4]
Oku dela K. Suzuki T. Umeda S. Tateishi Y. Mitsui H. Miyagi Y. Ohashi K. A comprehensive search for microRNAs with expression profiles modulated by oncogenic KRAS: Potential involvement of miR-31 in lung carcinogenesisOncol. [score:3]
Although many studies suggest that miR-31 is an oncogenic microRNA, there are reports suggesting that miR-31 functions as a tumor suppressor in lung cancer [95, 96]. [score:3]
Further research is required to determine whether miR-31 plays a pleiotropic role in individual tumors. [score:1]
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[+] score: 41
In conclusion, a more sensible and specific quantification of miRNAs by absolute Q-PCR analysis highlighted common up-regulation of miR-206, miR-223, miR-199a-5p, miR-199b*, miR-27a, miR-128a, miR-31 and miR-142-5p, and down-regulation of miR-17 in dystrophic fibres isolated from TA, DIA and VA of the adult mdx mouse (Figure S1). [score:7]
Importantly, up-regulation of miR-128a and miR-31 in dystrophic single muscle fibres is strongly supported by their recently discovered involvement in MDs and myogenesis [32], [33]. [score:4]
MiR-206, miR-31, miR-21, miR-335-5p, miR-27a, miR-142-5p and miR-223 were significantly up-regulated afterwards muscle damage respect damaged muscle. [score:4]
More importantly, miRNAs recently associated to muscle regeneration such as miR-31, miR-206 and miR-335-5p were confirmed over-expressed after acute damage [16]. [score:3]
In particular, some miRNAs decreased to control levels in old mdx mice (miR-15b, miR-17, miR-31 and miR-128a) (Figure 2B), suggesting a major involvement in compensatory mechanisms activated by the muscle in the early step of disease. [score:3]
Dystrophic muscle fibres isolated from different animal mo del of MDs were commonly characterized by the over -expression of several miRNAs (miR-15b, miR-21, miR-27a, miR-31, miR-128a, miR-142-5p, miR-199a-5p, miR-199b, miR199b*, miR-206, miR-221, miR-223 and miR-335-5p) with an expression profile strictly dependent on muscle impairment and damage accumulation (Figure 7). [score:3]
We identify fourteen miRNAs associated to dystrophic fibres (miR-15b, miR-17, miR-21, miR-27a, miR-31, miR-128a, miR-142-5p, miR-199a-5p, miR-199b, miR199b*, miR-206, miR-221, miR-223 and miR-335-5p) that may mediate muscle regeneration and remo delling in animal mo dels of MDs and acute muscle damage, and confirm over -expression of the previously identified regeneration -associated myomiR-206. [score:3]
Data obtained evidenced a group of miRNAs whose expression does not change during muscle repair afterwards acute damage (miR-15b, miR-17, miR-128a, miR-221, miR-199a-5p miR-199b and miR-199b*) (Table 1), and a group of miRNAs that are triggered afterwards CTX delivery (miR-206, miR-31, miR-21, miR-335-5p, miR-27a, miR-142-5p and miR-223) (Table 1), suggesting major involvement of the latter in muscle regeneration. [score:3]
Fourteen miRNAs were found dysregulated in dystrophic muscle fibres of the mdx mouse with differences linked to the originating muscle (miR-206, miR-199a-5p, miR-223, miR-199b, miR-199b*, miR-21, miR-221, miR-17, miR-15b, miR-31, miR-128a, miR-142-5p, miR-335-5p and miR-27a). [score:2]
In particular, the dysregulation was limited to miR-199b*, miR-31, miR-142-5p and miR-221 in dystrophic TPZ; to miR-128a, miR-21, miR-221 and miR-35-5p in dystrophic DIA; and to miR-15b, miR-17, miR-27a, miR-142-5p, miR-128a, miR-335-5p, miR-21, miR-31 in dystrophic VA (Figure 3B). [score:2]
In agreement with data already published characterizing the miRNome of mdx and DMD muscle [15], [16], [17], the over -expression of several miRNAs (miR-21, miR-31, miR-199a-5p, miR-199b, miR-142-5p, miR-221, miR-223 and miR-335-5p) was confirmed in murine dystrophic single muscle fibres. [score:1]
In support to this: miR-335 and miR-21 were found in human mesenchymal stromal cells [50] and in mesenchymal stem cells (MSCs) together with miR-21, miR-27a, miR-128a, miR-199b [51] miR-15b, miR-17, miR-21, miR-27a, miR-31, miR-199a, miR-199b, miR-221 and miR-335-5p were found in MSCs and in MSC secreted microparticles [49], [52], [53]. [score:1]
Otherwise a group of miRNAs recently correlated to myogenesis (miR-27a, miR-31, miR-221) [33], [37], [38] were confirmed to be mis-modulated in dystrophic single muscle fibres. [score:1]
MiR-15b, miR-17, miR-31, miR-128a, miR-142-5p and miR-335-3p were specifically induced in single muscle fibres of dystrophic DIA (Figure 1). [score:1]
This study demonstrated a common signature of DMD and ischemic muscle, outlining three different families of DMD-signature miRNAs: inflammatory (miR-222 and miR-223), degenerative (miR-1, miR-29c, and miR-135a) and regenerative (miR-31, miR-34c, myomiR-206, miR-335, miR-449, and miR-494). [score:1]
Only 7 of the 14 miRNAs associated to dystrophic fibres (miR-206, miR-31, miR-21, miR-335-5p, miR-27a, miR-142-5p and miR-223) were triggered by CTX injury. [score:1]
A more accurate and sensible quantification of miRNAs by Q-PCR analysis, extended the miRNA signature common to dystrophic single fibres of TA, DIA and VA to miR-128a, miR-31 and miR-142-5p (Figure S1). [score:1]
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[+] score: 41
Downregulation of dre-miR-126 can resulted in the upregulation of PI3KR2 and SPRED1, two negative regulators of the VEGF signaling pathway, upregulation of dre-miR-31 can lead to reduced venous sprouting. [score:11]
Of the miRNAs selected for comparison, two miRNAs (dre-miR-31 and dre-miR-430a) were up-regulated whereas two miRNAs (dre-miR-125a and dre-miR-126) were down-regulated based on the results of microarray analysis. [score:7]
Thus, in the present study, the upregulation of miR-31 and downregulation of miR-126 may be related to the vascular defects of embryos caused by (Figure 7). [score:7]
0022676.g007 Figure 7 caused aberrant expression of dre-miR-126 and dre-miR-31. [score:3]
caused aberrant expression of dre-miR-126 and dre-miR-31. [score:3]
Pedrioli et al. [26] observed that over -expression of miR-31 reduce venous sprouting of zebrafish embryo. [score:3]
Such as miRNA-126 and miRNA-31 that are demonstrated to be crucial for the vascular development in embryos of zebrafish. [score:2]
MiR-31 and miR-126, two miRNAs that have been proved to contribute to vascular development were significantly altered in the present study. [score:2]
Dre-miR-126 and dre-miR-31 pathways may together cause significantly decreased number of complete ISVs in zebrafish embryos. [score:1]
To validate the microarray data, we assayed expression levels of four miRNAs (dre-miR-125a, dre-miR-126, dre-miR-31, and dre-miR-430a) by qPCR and compared the results from the microarray and qPCR. [score:1]
It is possible that except miR-126 and miR-31 there are other known or unknown miRNAs contributing to the formation of ISVs. [score:1]
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39
[+] score: 41
a Over -expression of miR-31 restores chemo-response by reducing stathmin expression; miR-101/stathmin pathway contributes to radioresistance in human NPC; down-regulation of miR-193b promotes migration and proliferation of tumor cells by targets stathmin; miR-223 regulates stathmin by JNK signaling pathway to regulate MPM cell motility; b up-regulation of miR193b reduces proliferation and migration by inhibiting stathmin and uPA; silencing of miR-210 promotes proliferation of cancerous cells; transfection of miR-142 and miR-223 decreases expression of stathmin and IGF-1R to inhibit proliferation of cancerous cells; c microrna-9 inhibits cell proliferation, vasculogenic mimicry and tumor growth through controlling stathmin expression; miR-101 suppresses autophagy via targets stathmin and down-regulation of miR-101 is linked to the increase of cellular proliferation and invasiveness. [score:32]
Down-regulation of stathmin can partly renew taxane-sensitivity of KF-TX cells, and up-regulation of miR-31 can significantly recover chemo-sensitivity of KF-TX cells (KF-TX-miR-31) by reducing stathmin expression as well [69]. [score:9]
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40
[+] score: 41
Finally, linking differentially expressed miRNAs to their potential differentially expressed target using the algorithm published by Kertesz et al [25] identified a total of 11 potential miRNA-mRNA pairs: COL21A1 (collagen, type XXI, alpha 1; targeted by hsa-miR-155), CYP46A1 (cytochrome P450, family 46, subfamily A, polypeptide 1; targeted by hsa-miR-342-3p), KCNJ1 (potassium inwardly-rectifying channel, subfamily J, member 1; targeted by hsa-miR-155), MADCAM1 (mucosal vascular addressin cell adhesion molecule 1; targeted by hsa-let-7i), MRPS26 (mitochondrial ribosomal protein S26; targeted by hsa-miR-15a), OR2T29 (olfactory receptor, family 2, subfamily T, member 29; targeted by hsa-miR-143), RPS9 (ribosomal protein S9; targeted by hsa-miR-132), SLC10A1 (solute carrier family 10 (sodium/bile acid cotransporter family), member 1; targeted by hsa-miR-31), SLC16A8 (solute carrier family 16, member 8 (monocarboxylic acid transporter 3); targeted by hsa-miR-31), SNTG1 (syntrophin, gamma 1; targeted by hsa-miR-21) and TRPC5 (transient receptor potential cation channel, subfamily C, member 5; targeted by hsa-miR-335). [score:29]
In addition, miR-31, miR-155 and miR-21 were up-regulated in HNSCC [45], [50], which is also in concordance with our observation. [score:4]
They reported the up-regulation of miR-21, miR-155, miR-130b, miR-223 and miR-31, which is in concordance with our findings [45]. [score:4]
In contrast, blocking miR-31 expression reduced the growth of tumor xenografts [52]. [score:3]
Remarkably, it has been shown that the plasma miR-31 in patients with oral squamous cell carcinomas was reduced after tumor resection suggesting that this marker is tumor -associated [51]. [score:1]
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[+] score: 39
Elevated microRNA-31 expression regulates colorectal cancer progression by repressing its target gene SATB2. [score:6]
MicroRNA-31 contributes to colorectal cancer development by targeting factor inhibiting HIF-1alpha (FIH-1). [score:5]
The tumor suppressor gene RhoBTB1 is a novel target of miR-31 in human colon cancer. [score:5]
In contrast to Let-7, miR-31 appears to be a potent stimulator of KRAS in CRC, via negative regulation of RASA1, an inhibitor of KRAS function (Kent et al., 2016; Sun et al., 2013). [score:4]
More frequently, high miR-31 expression is associated with BRAF mutations and an aggressive cancer phenotype (Ito et al., 2014; Nosho et al., 2014; Choi et al., 2016). [score:4]
KRAS augments expression of miR-31. [score:3]
Altered expression of miR-21, miR-31, miR-143 and miR-145 is related to clinicopathologic features of colorectal cancer. [score:3]
MicroRNA-31 expression in relation to BRAF mutation, CpG island methylation and colorectal continuum in serrated lesions. [score:3]
Transcriptional regulation of miR-31 by oncogenic KRAS mediates metastatic phenotypes by repressing RASA1. [score:2]
Association of microRNA-31 with BRAF mutation, colorectal cancer survival and serrated pathway. [score:2]
Clinical relevance of microRNA miR-21, miR-31, miR-92a, miR-101, miR-106a and miR-145 in colorectal cancer. [score:1]
However, our present understanding of the molecular function of miR-31 is limited, although this oncomiR is reported to be transcriptionally activated downstream of the KRAS-BRAF-MAPK signaling cascade (Kent et al., 2016). [score:1]
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42
[+] score: 38
When analyzing the expression levels of the microRNAs selected for validation (Table 2), we observed that 5 of them (hsa-miR-135b *, hsa-miR-592, hsa-miR-31, hsa-mir-135b, hsa-miR-944) have similar patterns of expression, i. e., there are statistically significant differences between the UAs and the control group. [score:5]
Siow et al. [31] indicated that its overexpression would be related to the tumor stage, with a special relevance in the early stages, while others described [32] that the activation of the EGFR-AKT-CEBPb pathway would promote overexpression of miR-31 in oral cancer. [score:5]
Finally, it is important to mention that different authors [33– 35] described a direct association between the altered expression of miR-31 and the RAS-RAF-MAPK pathway, resulting in cell growth and cell survival, and which is closely associated with ameloblastoma pathogenesis, lung cancer [34] and colorectal cancer [33, 35]. [score:4]
Moreover, Sun et al. (2013) demonstrated that miR-31 can activate the RAS signaling pathway by inhibiting the RASA1 molecule, which would lead to increase cell growth and would facilitate tumorogenesis. [score:3]
On the other hand, Sun et al. (2013) detected a significant overexpression of miR-31 in colorectal cancer. [score:3]
On the SA group, we observed differences in the expression between SAs and the control group in seven out of the 13 microRNAs selected for biological validation (p <0.05), (hsa-miR-135b *, hsa-miR- 489, hsa-miR-592, hsa-miR-369-5p, hsa-miR-31, hsa-mir-135b, hsa-miR-944). [score:3]
When analyzing the expression of each miRNA, we observed statistically significant differences in 6 microRNAs among the UAs and the control group, (p <0.05) (hsa-miR-135b *, hsa-miR-592, hsa-miR-31, hsa-mir-135b, hsa-miR-944, hsa-miR-142-5p)). [score:3]
Edmonds et al. (2016) determined that miR-31 was not only highly overexpressed in lung adenocarcinoma but also significantly correlated with patient survival. [score:3]
In view of these results, we believe that miR-31 could be linked with the tumoral growth of the ameloblastoma and with the regulation of osteogenesis. [score:2]
MicroRNAs with a differential expression in both SA and UA when compared to the control group are the microRNA miR-135b, miR-135b *, miR-31, miR-592 and miR-944. [score:2]
In relation to miR-31, studies indicate that it would play an important role in the regulation of osteogenesis [29, 30]. [score:2]
After applying the inclusion criteria (|FC| <0.2 or> 5 and p adjusted <0.05), as previously mentioned, biological validation was performed by RT-qPCR of the 13 differently regulated miRNAs (hsa-miR-9, hsa-miR-135b*, hsa-miR-194*, hsa-miR-489, hsa-miR-592, hsa-miR-369-5p, hsa-miR-876-5p, hsa-miR-31, hsa-miR-135b, hsa-miR-211, hsa-miR-944, hsa-miR-142-5p, hsa-miR-455-3p), in an independent set of 46 samples corresponding to 19 SA, 8 UA and 19 controls. [score:2]
In addition, miR-31 would also participate in different neoplastic processes, including oral squamous cell carcinoma [31, 32]. [score:1]
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43
[+] score: 38
As reported by a previous study, miR-31 inhibits differentiation of naïve T cells to T regulatory cells by negatively regulating FOXP3 expression, binding directly to its potential target site in the 3′ UTR of FOXP3 mRNA. [score:10]
miR-31 was expressed uniquely in T cells (at similar levels in CD4 and CD8 T cells) and was 59.2 fold up-regulated in T cells vs NK cells, the next highest expressing cell type (p* = 9.9e-9). [score:8]
Consequently, miR-31 is down-regulated in nTreg compared to naïve CD4 [+]CD25 [−] T cells [30], permitting FOXP3 expression and establishment of T regulatory cell identity. [score:6]
miR-31 and miR-143 were verified to be specifically expressed in T cells and neutrophils in the single donor data (Figure S2). [score:3]
Four small -RNA transcripts were each found to be expressed specifically in one cell type: miR-378 in monocytes, miR-31 in T cells, miR-935 in eosinophils and miR-143 in neutrophils (Figure 1). [score:3]
Expression levels for miR-31, miR-143, miR-223 and miR-150 (Log2, mean ± SEM) are plotted across a panel of immune cell subsets, for samples obtained from single donors (dark shaded bars) and pooled donors (light shaded bars). [score:3]
Specificity of miR-31 to T cells appears to be consistent with its role in T cell lineage determination. [score:1]
Figure S2 miR-31, miR-143, miR-223 and miR-150 are confirmed to be cell type specific, using data from single donor samples. [score:1]
All miRNAs reported as specific to single cell types in the Roche dataset (miR-143, miR-31, miR-935 and miR-378) remained significant in the HUG dataset, while additional miRNAs were found to be cell-type specific, most notably miR-145 in neutrophils, miR-181 in NK cells and miR-146 in T cells and NK cells. [score:1]
We identified miR-143 as neutrophil specific, miR-31 as T-cell specific, miR-378 as monocyte specific and miR-935 as eosinophil specific. [score:1]
A) miR-143 and miR-31 were specific to neutrophils and T cells respectively, while B) miR-362 and miR-125 were specific to monocytes, pDCs and T cells, neutrophils. [score:1]
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[+] score: 37
In order to investigate whether miR-31 regulation of these target genes affects metastatic function, the six identified target genes were reexpressed in miR-31 -expressing cells. [score:8]
The miR-31, which is known to have pleiotropic effects on breast cancer metastasis, has been shown to inhibit metastasis at multiple steps by inhibiting the expression of prometastatic genes [81]. [score:7]
These findings indicate that miR-31 inhibits metastasis, which suggests that it may be an ideal therapeutic target for breast cancer. [score:5]
Investigations that explored miR-31 expression in MDA-MB-231 and SUM-159 breast cancer cells found that ectopic expression of miR-31 in vivo and in vitro hindered invasion and metastatic colonization [81]. [score:3]
The experiment showed that even though overexpressing miR-31 in breast cancer cells resulted in larger tumors and increased proliferation, the cancers were encapsulated and less invasive. [score:3]
In accordance, inhibiting miR-31 resulted in increased invasiveness and metastasis in vivo. [score:3]
Results showed that only ITGA5, RDX, and RhoA could reverse the motility defects and impaired invasion, which suggests that these three genes are important targets of miR-31. [score:3]
The miR-31 may regulate at least 200 different mRNAs in mammalian cells. [score:2]
3.7. miRNA-31. [score:1]
Normal levels are exhibited in normal breast epithelial cells, but miR-31 is almost undetectable in metastatic breast cancer cells in vivo. [score:1]
Furthermore, miR-31 also reduces cell survival and the ability to form secondary tumors. [score:1]
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45
[+] score: 35
In contrast, knocking down both miR-21 and miR-31 inhibited K15M -mediated cell motility, which indicated that targeting K15 or its downstream-regulated microRNAs may represent novel therapies for treatment of KSHV -associated neoplasia [22]. [score:7]
One of the mechanisms identified for the miR-31 mediated increasing cell motility was through direct repression of a novel tumor suppressor and inhibitor of migration, FAT4; moreover, a reduction of FAT4 enhanced EC mobility [23]. [score:6]
In addition, Lagos et al. reported two groups of cellular miRNAs induced during primary KSHV infection of LECs: the “early” group reached its peak of expression at six hours post-infection, and included miR-146a, miR-31 and miR-132; the “late” group, which included miR-193a and Let-7i, steadily increased its expression during the next 72 hours [48]. [score:5]
Interestingly, these miRNAs (miR-21, miR-31, miR-221/222, miR-30) can act as either “oncogenes” or “tumor-suppressor genes” in a variety of cancers in which they can regulate tumor cell proliferation, apoptosis, invasion, angiogenesis, metastasis and other important cellular functions [26, 27, 28, 29, 30], indicating functional relevance of these regulatory miRNAs in virus-related malignancies. [score:5]
Human miRNAs Validated Targets Regulated by Viral Proteins Functions References miR-21 - K15M Cell mobility[22] miR-31 FAT4 K15M Cell mobility[22, 23] miR-221/222 ETS2/ETS1 LANA and Kaposin B Cell migration[23] miR-30b/c DLL4 - Angiogenesis[25] miR-557/766/1227/1258/1301 RTA - Viral replication[39] miR-146a CXCR4 vFLIP Immune response[41] miR-1293 vIL-6 ORF57 Immune response[46, 47] miR-608 hIL-6 ORF57 Immune response[46, 47] miR-132 p300 - Immune escape[48] This work was supported by grants from a Center for Biomedical Research Excellence P20-GM103501 subaward (RR021970), the Ladies Leukemia League Grant (2014-2015), and the National Natural Science Foundation (NNSF) of China (81101791, 81272191, 81472547 and 81400164). [score:4]
Upregulation of miR-31 by KSHV was further confirmed in virally infected lymphatic endothelial cells (LECs), in which depletion of miR-31 reduced cell mobility [23]. [score:4]
Tsai and colleagues reported that KSHV-encoded K15 protein, minor form (K15M), can induce cell migration and invasion, potentially through upregulation of cellular miR-21 and miR-31 via its conserved Src-Homology 2 (SH2) -binding motif [22]. [score:4]
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Knockdown of SMAD4 by siRNA downregulated the expression of miR-31 and miR-181a indicating a possible involvement of BMP2 as a negative regulator of LEC identity (51). [score:8]
In addition, KSHV can influence endothelial cell motility by downregulating the miR-221/miR-222 cluster and upregulating miR-31 (93). [score:7]
Whether upregulation of miR-31 can regulate PROX1 during KSHV infection is unknown. [score:5]
Lymphatic identity is maintained through suppression of the blood endothelial cell (BEC)-enriched miRNAs miR-31 and miR-181a, which can repress LEC-specific genes, including the master LEC fate regulator PROX1 and the receptor tyrosine kinase vascular endothelial growth factor receptor-3 (VEGFR-3). [score:4]
Interestingly, BMP2 signaling upregulated miRNAs: miR-194, miR-186, miR-99a, miR-92a and also miR-31, and miR-181a (51). [score:4]
Both miR-31 and miR-181a can target PROX1 and as a result repress LEC-specific genes, including VEGFR-3, and vascular development in embryonic LECs (22, 40). [score:4]
Overexpression of miR-31 was shown to repress FOXC2 and several other LEC-signature genes (40). [score:3]
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Interestingly, miR-31 has been shown to function as a negative regulator of osteogenesis and was down-regulated in MSCs during osteogenic differentiation [15, 31, 52– 54]. [score:5]
Among the differentially expressed microRNAs in Exo_D21 versus Exo_P6, five microRNAs (miR-31-3p, miR-31-5p, miR-29b-3p, miR-148b-3p and miR-29c-3p) were decreased two-fold or more, while only one microRNA (miR-10b-5p) had more than a two-fold increase in expression. [score:5]
Moreover, microvesicular miR-31 derived from senescent endothelial cells inhibits the osteogenic differentiation of MSCs by down -regulating receptors of Wnt protein FZD3 [55]. [score:4]
Moreover, five microRNAs (miR-31-3p, miR-29b-3p, miR-29c-3p, miR-10b-5p and miR-27b-3p) had significant changes in expression in Exo_D21 compared with their expression in both Exo_P6 and Exo_D3. [score:4]
Taken together, the lower expression of exosomal miR-31, miR-221 and miR-144 from the late stage of differentiation may contribute to the induction of osteogenic differentiation, whereas the lower osteogenic differentiation of cells treated by exosomes from expansion or early osteogenic differentiation may partly be due to the delivery of higher exosomal miR-31, miR-221 and miR-144. [score:3]
It is worth noting that, among the differentially expressed exosomal microRNAs, miR-31 was dramatically decreased in exosomes from the late stage of osteogenic differentiation. [score:3]
The inhibition of miR-31 in MSCs improves the repair of bone defects in vivo indicated by increased bone volume and bone mineral density [31, 53]. [score:3]
MiR-31 has been reported to inhibit osteogenic differentiation via the down regulation of osteogenic transcription factors Osterix (Osx) and special AT-rich sequence -binding protein 2 (SATB2), which are both downstream of RUNX2 [15, 52]. [score:3]
Interestingly, the two highest regulated microRNAs in Exo_D21 versus Exo_D3 and Exo_P6, miR-31-3p/5p and miR-10b-5p, are involved in osteogenic differentiation and cell recruitment [30, 31]. [score:2]
However, when comparing Exo_D21 with Exo_D3, three microRNAs (miR-31-3p, miR-144-3p and miR-29c-3p) were reduced more than two-fold and two microRNAs (miR-154-5p and miR-10b-5p) were increased more than two-fold. [score:1]
This provides evidence that exosomal miR-31 is biologically active and can be transferred to the recipient cells to exert their function. [score:1]
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Two of them, miR-133b and miR-145 were down-regulated and the remaining four, miR-31, miR-96, miR-135b and miR-183, were up-regulated, suggesting that they may potentially act as tumor suppressor genes or oncogenes, respectively. [score:9]
For the up-regulated miRNAs, miR-135b, miR-31, miR-96 and miR-183, it may be expected that gene targets belong to the class of tumor suppressor genes. [score:8]
Among the differentially expressed miRNAs, miR-31, miR-96, miR-133b, miR-135b, miR-145 and miR-183 as the most consistently deregulated in CRC. [score:4]
The up-regulation of miR-31 was significantly higher in stage IV than in CRC samples stage II (p = 0.028) (Figure 4). [score:4]
Figure 4 Real-time PCR analysis of miR-31 expression between stage II and stage IV tumor samples. [score:3]
Other members of the forkhead family transcription factors, such as FOXC2 and FOXP3, were identified as putative targets of miR-31. [score:3]
The expression levels of miR-31 were higher in the tumor samples and CRC cell lines in comparison to the non-tumoral samples and was related to pathological stage, suggesting that this miRNA could contribute to both, the tumorogenesis and the acquisition of a more aggressive phenotype in CRC. [score:3]
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49
[+] score: 33
Other miRNAs from this paper: hsa-mir-188, oar-mir-3957
We first performed miRNA and gene expression analysis in sheep skin and SFFs, and the results showed that miR-188, miR-3957-5p, miR-31 and DLX3 were to some extent expressed in both sheep skin and SFFs (Fig 2A), implying that it is possible that miR-188 and miR-3957-5p regulate sheep DLX3 gene posttranscriptionally. [score:6]
MiR-31 and miR-188 downregulate the gene expression of DLX3, HOXC13, GATA3, KRT71 and LEF1 mRNA in SFFs. [score:6]
Of these miRNAs, miR-31 has been identified to target DLX3, fibroblast growth factor 10 (FGF10), keratin 16 (KRT16) and keratin 17 (KRT17) genes, and regulates hair follicle development and hair fibre formation [8]. [score:5]
The results showed that, similar to miR-31, miR-188 regulated the expression of these genes (HOXC13, GATA3, KRT71 and LEF1) in SFFs (S2B–S2E Fig). [score:4]
The real-time RT-PCR expression analysis showed that, similar to miR-31, in agreement with reporter gene findings, miR-188 mimc reduced the endogenous DLX3 gene expression in SFFs, compared to the mimics negative control (S2A Fig), which is in agreement with reporter gene findings. [score:4]
MiR-31 has been shown to target DLX3 in hair follicle [8] and none of the four studied SNPs are locate within the miR-31 binding site. [score:3]
The following primers were used for miRNA expression analysis: miR-31: 5′-CAGGCAAGATGCTGGCATAGCT-3′; miR-188: 5′-ATCCCTTGCATGGTGGAGGGT-3′. [score:2]
About >30% suppression in the reporter activity was observed, compared to the control (cells cotransfected with psiCHECK2 vector and miR-31 mimic). [score:2]
The reporter gene assay showed that as expected, miR-31 mimic could cause > 30% suppression of the luciferase activities of the 3′UTR luciferase reporters (psiCHECK2-CCGI, psiCHECK2-TTAD or psiCHECK2-TCAD) in both SFFs and HaCaT cells, compared to the negative control (Fig 2B), consistent with the previous report in mice [8]. [score:1]
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50
[+] score: 33
The upregulation of miR-31 in T cells may correlate with the deficiency of Treg cell development/function in lupus since miR-31 targets Foxp3, a critical transcription factor for Treg cell development and function [32]. [score:8]
Impressively, the expression levels of miR-182-96-183, miR-31, and miR-155 were markedly upregulated in 9-month old NZB/W mice when compared to either 9-month old NZW or 3-4-month old NZB/W mice (Fig. 4B). [score:5]
As shown in the figure 5, the expression levels of selected lupus -associated miRNAs including miR-96, miR-31, miR-127, miR-146a and miR-155 were significantly upregulated in freshly-isolated splenocytes from 3-4-month old MRL-lpr mice when compared to 1-month old MRL-lpr mice. [score:5]
0014302.g005 Figure 5The expression levels of selected lupus -associated miRNAs (miR-96, miR-31, miR-127, miR-146a, and miR-155) in freshly-isolated (MRL-lpr-1 month), 24 hrs of LPS activated (MRL-lpr-1 month-LPS) splenocytes from 1-month old MRL-lpr mice, and freshly isolated splenocytes from 3-4 month old MRL-lpr mice (MRL-lpr-3-4 months) were analyzed by Real-time RT-PCR. [score:3]
The expression levels of selected lupus -associated miRNAs (miR-96, miR-31, miR-127, miR-146a, and miR-155) in freshly-isolated (MRL-lpr-1 month), 24 hrs of LPS activated (MRL-lpr-1 month-LPS) splenocytes from 1-month old MRL-lpr mice, and freshly isolated splenocytes from 3-4 month old MRL-lpr mice (MRL-lpr-3-4 months) were analyzed by Real-time RT-PCR. [score:3]
Overall, our data revealed that the expression changes in lupus -associated miRNAs such as miR-182-96-183, miR-31, miR-127, miR-379, miR-155, and miR-150 that were observed in splenocytes were also evident in purified splenic B and T cells. [score:3]
However, LPS activation did not induce miR-96, miR-31 and miR-127 expression changes in splenocytes. [score:3]
Consistent with the data observed in whole splenocytes, the expression of miR-182-96-183, miR-31, miR-127 and miR-379 was also significantly increased in purified splenic B cells and T cells from MRL-lpr mice when compared to MRL mice (Fig. 2B). [score:2]
Despite the genetic differences among these three lupus stains, a common set of dysregulated miRNAs (miR-182-96-183 cluster, miR-31, and miR-155) was identified in splenocytes when compared with age-matched control mice. [score:1]
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51
[+] score: 32
When we compared the mRNA and miRNA profiles, differentially regulated in PKD, with Argonaute (a comprehensive database on miRNAs; [45, 71]), there were few genes reported as miRNA target like tropomyosin 1, alpha (TPM1) as a target of miR-21, the beta polypeptide of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAB), regulatory subunit 9B of protein phosphatase 1 (PPP1R9B), early growth response 3 (EGR3) and dynamin 1-like (DNM1L) as targets of miR-31, plysia ras-related homolog A2 (RHOA) as targets of miR-217, etc. [score:10]
miRNA Target Genes Pathways miR-128 ABCB9, BTG1, DSCR1, RASD1 ABC transporters General miR-136 GRN, PPP1R9B miR-147 HOXA1, PTGFRN miR-148 EGR3, SCN3A miR-181b IGF1R, NKX6-1 Adherens junction, Maturity onset diabetes of the, Focal adhesion, **Long term depression miR-196a ABCB9, CPB2, IRS1, MAPK10 ABC transporters General, Complement and coagulation cas, Adipocytokine signaling pathwa, Insulin signaling pathway, Type II diabetes mellitus, Fc epsilon RI signaling pathwa, Focal adhesion, **GnRH signaling pathway, **MAPK signaling pathway, Toll like receptor signaling p, Wnt signaling pathway miR-203 SARA1 miR-20 BTG1, SARA1, YWHAB Cell cycle miR-21 TPM1 mir-216 GNAZ **Long term depression miR-217 RHOA Adherens junction, Axon guidance, Focal adhesion, Leukocyte transendothelial mig, Regulation of actin cytoskelet, TGF beta signaling pathway, T cell receptor signaling path, Tight junction, Wnt signaling pathway miR-31 ATP2B2, DNM1L, EGR3, PPP1R9B, YWHAB **Calcium signaling pathway, Cell cycle miR-7 SLC23A2 miR-7b HRH3, NCDN, SLC23A2 **Neuroactive ligand receptor in b: miRNAs and their targets (from TargetScan and miRanda). [score:8]
In line with the expression on the miRNA-arrays, in qPCR analysis (Figure 4), miR-31 was 3.15 fold down regulated in diseased samples compared to healthy tissue. [score:5]
We predict that several of the differentially regulated genes are miRNA targets and miR-21, miR-31, miR-128, miR-147 and miR-217 may be the important players in such interaction. [score:4]
Interestingly, the expressions of miR-31 and miR-217 have not been previously reported in kidney. [score:3]
It is interesting to note that miR-31 and miR-217 have not been previously reported in kidney. [score:1]
Furthermore, we describe some newly detected miRNAs, miR-31 and miR-217, in the kidney which have not been reported previously. [score:1]
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52
[+] score: 32
The miRNA expression patterns in clinical specimens directly shows that most candidate Suppressive miRNAs, except for miR-31, are expressed at significantly lower levels than the candidate Oncogenic miR-17 (Figure 3). [score:8]
The mean expression level of most candidate Suppressive miRNAs in the clinical specimens was between the corresponding miRNA values in the PAG and HAG cells, except for miR-185 and miR-31. [score:5]
As expected, a considerable variability in miRNA expression was observed among the individual specimens, mainly of miR-31 (Figure 3). [score:3]
miR-31 was reported to exert inhibitory effects on metastasis in breast cancer [33], [34] and in mesothelioma [17], while it has a potential oncogenic role in as head and neck squamous cell carcinoma and lung cancer [35], [36]. [score:3]
Three miRNAs were within expression range (miR-34a, miR-185 and miR-204, Figure 2C) and two which were silenced (miR-31 and miR-184, Figure 2B) in the HAG cells. [score:3]
Our results concur with the suggested inhibitory role for miR-34a and miR-31. [score:3]
Tube formation activity was substantially inhibited by miR-34a and miR-185, and more mildly by miR-31 and miR-184, but not by miR-204, as compared to control (Figure 5, A–F). [score:2]
Remarkably, a substantial and consistent inhibition in net proliferation was conferred by miR-31, miR-34a, miR-184 and miR-185 as compared to the control cell (Figure 4B). [score:2]
Different roles of miR-17, miR-31 and miR-34a in cancer have been reported before, although never in cutaneous melanoma. [score:1]
While the mean level of miR-185 was very close to the PAG cells, miR-31 levels were clearly higher even than PAG cells (Figure 3). [score:1]
In some cases, cancer is facilitated by the loss of certain miRNAs, such as miR-15/16 cluster in chronic lymphocytic leukemia [15], miR-34a in uveal melanoma [16] and miR-31 in mesothelioma [17]. [score:1]
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[+] score: 32
Hsa-miR-31 has been found dysregulated in several primary epithelial tumors where, according to tumor localization, it seems to trigger different cancer pathways: it is up-regulated in some carcinomas [36]– [41], and down-regulated in others [42]– [45]. [score:8]
A weaker staining was observed in tumor and normal colonic cells, but only few hsa-miR-31 expressing lymphocytes were present in normal lymph nodes, suggesting that hsa-miR-31 upregulation resulted mostly from the inflammatory rather than from the malignant component of the tumor. [score:6]
Targeted functions of hsa-miR-31 include angiogenesis, where its upregulation increases blood, but not lymphatic vessels sprouting [48], and acute inflammation [49]. [score:6]
We found that hsa-miR-31 is mostly up-regulated in peritumoral inflammatory cells, compared to cancer and normal cells. [score:3]
Such a duality, has been found in invasive tumors as well: in colon cancer cell lines, invasion is promoted by hsa-miR-31 overexpression [46], [47], while, in breast cancer cell lines, its low levels promote a more aggressive behavior [42], which correlates to a poorer patient prognosis. [score:3]
Thus, hsa-miR-31 dysregulation in primary aggressive tumors is an indisputable evidence, but the interplay of its functions between the inflammatory, neoangiogenic and tumoral metastatic context, needs, still, to be elucidated. [score:2]
With the exception of hsa-miR-31, in situ hybridization (ISH) of colonic adenocarcinomas and the corresponding adjacent normal tissue, as well as its nodal and liver metastasis tissue microarrays, confirmed the signatures identified by the cards and qRTPCR analyses. [score:1]
Interestingly, hsa-miR-31 ISH (data not shown) revealed a very strong signal in inflammatory cells surrounding the tumors. [score:1]
0096670.g001 Figure 1Normal colon tissue (NORM), colon adenocarcinomas (ADENOCA), lymphonodal metastasis (POSLYM) and colon liver (LIVERMET) for miR-21(A), miR-31 (B), miR-93 (C), miR-103 (D) and miR-566 (E). [score:1]
Normal colon tissue (NORM), colon adenocarcinomas (ADENOCA), lymphonodal metastasis (POSLYM) and colon liver (LIVERMET) for miR-21(A), miR-31 (B), miR-93 (C), miR-103 (D) and miR-566 (E). [score:1]
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[+] score: 32
In silico target prediction of selected miRNAs, such as miR-31 and miR-4417 which are upregulated in tumor samples [57, 58], revealed a set of potential target mRNAs including Flavin containing monooxigenase 4 (FMO4), cyclin dependent kinase inhibitor 2B (CDKN2B), and prostaglandin D2 receptor (PTGDR), whose expression pattern changed oppositely  according to our (Human Transcriptome Array 2.0 microarray) results. [score:12]
Twelve were upregulated (miR-31, 8-fold p < 0.001) and 11 were downregulated (miR-10b 3-fold p < 0.001) in neoplastic lesions compared to normal group. [score:6]
The highest miRNA expression alteration was observed in case of miR-31 showing eightfold higher expression both in adenoma and in CRC tissue compared to normal samples (Additional file 2: Table S2). [score:4]
FMO4 catalyzes NADPH -dependent oxidative metabolism and is downregulated in carcinoma cells by miR-31 [59]. [score:4]
Three miRNAs (miR-31, miR-4506, miR-452*) were differentially expressed in adenoma compared to normal both in tissue and plasma samples. [score:2]
Three miRNAs were selected: miR-31 has the highest fold change in normal vs. [score:1]
Three miRNA were selected: miR-31 has the highest fold change in normal vs. [score:1]
FMO4 is influenced by miR-31. [score:1]
These miRNAs could be abundant and are less affected by tissue processing (miR-135b, miR-196b, miR-31). [score:1]
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55
[+] score: 32
We could confirm the upregulation of 2 miRNAs (hsa-miR-31-5p and hsa-miR-223-3p) out of 9 miRNAs that were identified as dysregulated in active UC mucosa by Fasseu et al. Lin et al. identified 4 miRNAs with an increased expression in UC, of which we could confirm the upregulation of hsa-miR-31-5p and hsa-miR-146a-5p. [score:10]
All 6 miRNAs (hsa-miR-21-5p, hsa-miR-31-5p, hsa-146a-5p, hsa-miR-155-5p, hsa-miR-375 and hsa-miR-650) with increased expression in active UC demonstrated a similar upregulated expression profile in both active CDc and IC compared to healthy controls. [score:7]
The miRNA microarray expression data was confirmed by performing qRT-PCR for hsa-miR-200c-3p and 10 other differentially expressed miRNAs selected because of their highly significant p-value or fold change (hsa-miR-21-5p, hsa-miR-31-5p, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-miR-196b-5p, hsa-miR-196b-3p, hsa-miR-200b-3p, hsa-miR-375, hsa-miR-422a and hsa-miR-650). [score:5]
Here, we confirm the strong increase in expression of hsa-miR-31-5p in active UC vs. [score:3]
The same authors also reported a linear increase in expression of hsa-miR-31-5p along the evolution of normal colon tissue, to IBD and to IBD -associated dysplasia [39]. [score:3]
Our study can't confirm this hypothesis because expression levels of hsa-miR-31-5p were elevated in both UC and IC patients compared to controls. [score:2]
Although, miRNAs such as hsa-miR-21-5p, hsa-miR-31-5p or hsa-miR-155-5p are reported in several studies, and have potential as biomarker. [score:1]
The authors propose hsa-miR-31-5p as a diagnostic biomarker of IBD to differentiate from its mimics, including IC [19]. [score:1]
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[+] score: 30
RT-PCR validation of mostly dysregulated miRs confirmed that miR-138, miR-147b, miR-148a, miR-99a, miR-455-3p and miR-125b were significantly upregulated and miR-31-star, miR-422a, miR-330-3p, mir-330-5p and miR-378d were downregulated in PANC-1-GR cell clones vs. [score:8]
We suggest that under acquired chemoresistance, accumulation of mutant p53 induces expression of miR-21-5p, miR-31*, miR-125b-5p, miR-210-3p, miR-330-3p, miR-378a-3p, miR-422a and miR-486-5p which in turn enhances proliferation by upregulating Bcl-2 expression in PDAC cells. [score:8]
MiR-screening revealed significantly upregulated (miR-21, miR-99a, miR-100, miR-125b, miR-138, miR-210) and downregulated miRs (miR-31*, miR-330, miR-378) in chemoresistant PDAC (p<0.05). [score:7]
In MIA-PaCa-2-GR cell clones miR-125b, miR-210, miR-21, miR-100, miR-148a, miR-99a and miR-455-3p were significantly upregulated, whereas miR-330-3p, miR-330-5p, miR-486-5p, miR-422a and miR-31-star were significantly downregulated (Fig 6B). [score:7]
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57
[+] score: 30
Six miRNAs showed significant differential expression between early and late stage (mostly stage III) disease including an increase in miR-31 expression (Table 3). [score:7]
Subsequently, a group of 13 miRNAs showing differential expression in CRC tumors was identified with the expression level of miR-31 being correlated with CRC tumor stage [22]. [score:5]
MiR-31, miR-21 and members of the miR-17-92 cluster, and its paralogues, were shown to be up-regulated in CRC. [score:4]
The increase in miR-31 was reported previously [22], but was not found in several other miRNA expression studies in CRC [23, 26, 27, 35, 36]. [score:3]
We demonstrate that several miRNAs including miR-1 and miR-31 have evolving expression patterns between stages II and III of CRC and may provide potential prognostic or diagnostic markers for this cancer. [score:3]
miRNA fold change location CRC Fragile site* hsa-miR-20a up 13q31.3 gain hsa-miR-19a up 13q31.3 gain hsa-miR-17-5p up 13q31.3 gain hsa-miR-93 up 7q22.1 gain hsa-miR-25 up 7q22.1 gain hsa-miR-31 up 9p21.3 hsa-miR-106a up Xq26.2 hsa-miR-143 down 5q32 loss hsa-miR-145 down 5q32 loss hsa-miR-125a down 19q13.41 gain hsa-miR-1 down 18q12.3 or 20q13.33 loss (18q) or gain (20q)* Summarized from [41- 43] Figure 4 Expression of 14 miRNAs in 8 CRC cell lines and normal colon total RNA. [score:3]
miRNA fold change location CRC Fragile site* hsa-miR-20a up 13q31.3 gain hsa-miR-19a up 13q31.3 gain hsa-miR-17-5p up 13q31.3 gain hsa-miR-93 up 7q22.1 gain hsa-miR-25 up 7q22.1 gain hsa-miR-31 up 9p21.3 hsa-miR-106a up Xq26.2 hsa-miR-143 down 5q32 loss hsa-miR-145 down 5q32 loss hsa-miR-125a down 19q13.41 gain hsa-miR-1 down 18q12.3 or 20q13.33 loss (18q) or gain (20q)* Summarized from [41- 43] Figure 4 Expression of 14 miRNAs in 8 CRC cell lines and normal colon total RNA. [score:3]
However, the commonly deregulated miRNAs included miR-31, members of the miR-17-92 cluster, miR-1, miR-143 and miR145, thereby validating the findings of previous studies. [score:2]
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[+] score: 30
Liu et al. demonstrated that ectopic expression of miR-31 repressed its target factor-inhibiting hypoxia-inducible factor (FIH) expression to activate hypoxia-inducible factor (HIF) under normoxic conditions, both in vitro and in vivo. [score:9]
These miR-31 [∗] regulated functional effects were mediated by the regulation of fibroblast growth factor 3 (FGF3) [29] and RhoA [32] expression levels. [score:5]
The high salivary level was significantly reduced after excision of OSCC lesion, indicating that the main contributor for miR-31 upregulation was OSCC lesion [72]. [score:4]
miR-31 and its passenger strand miRNA (miR-31 [∗]) have been shown to be upregulated in oral leukoplakia (OLP) and OSCC and to have an oncogenic role in OSCC tumorigenesis [28– 31]. [score:4]
Additionally, miR-31-FIH-HIF-VEGF regulatory cascade was found to affect several biological processes such as cell proliferation, migration, and epithelial-mesenchymal transition (EMT) in OSCC cells [28]. [score:2]
Similarly, miR-31 [∗] regulated apoptosis, cell proliferation, migration, and invasion in OSCC cells [29]. [score:2]
Expression level of miR-31 in saliva was found to be significantly increased in patients with OSCC of all clinical stages as compared to that of the healthy controls. [score:2]
miR-31, miR-17/20a, miR-125b, miR-155, miR-181, miR-375 and miR-491-5p, miR-205, and miR-let7d were found to be associated with lymph node metastasis and poor OSCC patient survival [38, 59, 60, 65, 72, 77– 80]. [score:1]
Moreover, miR-31 was shown to collaborate with human telomerase reverse transcriptase (hTERT) to immortalize normal oral keratinocytes (NOKs), indicating that it might contribute to early stage oral carcinogenesis [31]. [score:1]
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59
[+] score: 29
Therefore, diminished expression of miR-31 post-wounding in aged skin may suppress keratinocyte proliferation. [score:5]
TGF-β inducible miR-132 and miR-31 were found to be upregulated during the transition from the inflammatory to the proliferative phase in human skin promoting keratinocyte proliferation 23, 28. [score:4]
miR-31 is known to be highly expressed in the activated keratinocytes under hyperproliferative conditions, including anagen phase of the hair cycle, psoriasis, and cutaneous squamous cell carcinoma 31– 35. [score:3]
Significantly increased levels of miR-31 in unwounded aged versus (vs) young skin were decreased on days 3 and 5 after injury, in contrast to increase in miR-31 expression in young mice on days 3 and 5 post-wounding (Fig.   1b, Supplementary Tables  1 and 2). [score:3]
miR-31 expression has been shown to be gradually increased in the epithelial tongue and promotes keratinocyte proliferation and migration during wound repair [28]. [score:3]
Interestingly, microarray validation by RT-qPCR confirmed the contrasting expression of miR-31. [score:3]
In conclusion, our study for the first time 1) reports differences in the expression of miRNAs in young and aged mouse skin wounds that suggest involvement of various miRNAs in age -associated impairment in wound healing; 2) provides evidence about contribution of miR-31 to the delay in wound healing in aged skin; 3) identifies miR-200c as an important player in successful re-epithelialisation during cutaneous wound healing that can exert positive and negative effects on keratinocyte differentiation and migration, respectively. [score:3]
In addition, the observed decreased miR-31 levels in aged skin may also contribute to the changes in the inflammatory response, as this miRNA has been found to positively regulate inflammatory cytokine and chemokine production in primary human keratinocytes [35]. [score:2]
Some notable examples include miR-130a, miR-132, miR-155, miR-198, miR-21, miR-31 and miR-378a 13, 23, 24, 26, 28– 30. miR-155 acts as an important player in controlling the inflammatory response during skin repair; genetic deletion of miR-155 in mice leads to accelerated healing associated with elevated numbers of macrophages and increased type-1 collagen deposition in wounded tissue [30]. [score:1]
Moreover, our finding reconciles well with the previous reports about the positive impact of miR-31 on re-epithelialisation during acute wound healing [28]. [score:1]
The observed changes in the dynamics of miR-31 levels in aged skin during wound healing suggest that miR-31 may compromise wound repair in aged skin. [score:1]
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60
[+] score: 28
MiR-31 can negatively regulate FOXP3 expression by binding directly to its potential target site in the 3′UTR of FOXP3 mRNA [92]. [score:6]
The inverse correlation between miR-31 expression and Treg cell number in the PBMC of H1N1 critically ill patients can be explained by the negative regulation of FOXP3 expression. [score:6]
P38 MAPKs (MAPK11, MAPK13, and MAPK14) were found to be regulated by miR-769-5p, miR-146b-5p, let-7g, miR-30b, miR-31, miR-361-3p, and miR-362-3p (Figure 7), which were all down expressed in H1N1 critically ill patients. [score:4]
validation of differentially expressed miRNAs and ROC analysisThe microarray data were validated by performing, qRT-PCR for nine miRNAs, including hsa-miR-146b-5p, hsa-miR-148a, hsa-miR-150, hsa-miR-31, hsa-miR-155, hsa-miR-29a, hsa-miR-29b, hsa-miR-342-5p, and hsa-miR-886-3p. [score:3]
Our miRNA microarray and RT-PCR analysis revealed that miR-31 was significantly down-expressed in PBMCs of H1N1 critically ill patients. [score:3]
The expression of hsa-miR-150, hsa-miR-31, hsa-miR-155, hsa-miR-29a, hsa-miR-29b, hsa-miR-342-5p, and hsa-miR-146b-5p were present in lower abundance, whereas hsa-miR-148a and hsa-miR-886-3p were present in higher abundance in PBMCs from critically ill patients infected with H1N1 influenza virus than that from healthy controls. [score:3]
The microarray data were validated by performing, qRT-PCR for nine miRNAs, including hsa-miR-146b-5p, hsa-miR-148a, hsa-miR-150, hsa-miR-31, hsa-miR-155, hsa-miR-29a, hsa-miR-29b, hsa-miR-342-5p, and hsa-miR-886-3p. [score:1]
ROC curve analyses revealed that miR-31, miR-29a and miR-148a were valuable biomarkers for differentiating critically ill patients from controls: miR-31 yielded an AUC (the areas under the ROC curve) of 0.9510 (95% CI: 0.8734–1.029; P = 0.0001884) with 81.82% sensitivity and 92.31% specificity in discriminating critically ill patients; miR-29a yielded AUC of 0.8951 (95% CI: 0.7412–1.049 P = 0.0001070) with 90.91% sensitivity and 92.31% specificity in discriminating critically ill patients, and miR-148a yielded AUC of 0.8811 (95% CI: 0.7360–1.026 P = 0.001601) with 72.73% sensitivity and 100% specificity in discriminating critically ill patients(Figure 5). [score:1]
ROC curve analyses revealed that miR-31, miR-29a and miR-148a all had significant potential diagnostic value for critically ill patients infected with H1N1 influenza virus, which yielded AUC of 0.9510, 0.8951 and 0.8811, respectively. [score:1]
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[+] score: 27
It has been demonstrated that miR-21 acts as a positive indirect regulator of FOXP3 expression; in contrast, miR-31 negatively regulates FOXP3 expression by binding directly to its target site in the 3′UTR of FOXP3 mRNA. [score:11]
Comparing miRNA expression profiles between human naïve CD4 [+] T cells with Tregs, miR-31 was found to be down-regulated in Treg cells, while miR-21 were found to be significantly up-regulated in this population [32]. [score:9]
miR-31 was significantly down-regulated in a few donors (p < 0.05; Additional file 1: Figure S2). [score:4]
miR-21 expression was induced after both antibody treatments, while miR-31 was consistently repressed by OKT3 treatment, and FvFcR treatment led to a variable response. [score:3]
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[+] score: 27
Focusing on the conserved miRNAs presented in Table 1, we found that of the 14 miRNAs downregulated in our study relative to normal bone, six were published as upregulated in osteosarcoma relative to osteoblasts, namely the miRNAs miR-126, miR-142-3p, miR-195, miR-223, miR-451 and miR-497, while miR-31/miR-31* was upregulated compared to bone and downregulated compared to osteoblasts. [score:11]
miR-451 and miR-497 showed a trend towards being significantly decreased, miR-31 showed a heterogenous expression pattern, and miR-19b, miR-29b and miR-142-3p were expressed at comparable level in clinical samples and bone. [score:5]
Furthermore, the upregulated miRNAs included miR-9/miR-9*, miR-21*, miR-31/miR-31*, miR-196a/miR-196b, miR-374a and members of the miR-29 and miR-130/301 families. [score:4]
Furthermore, the overexpressed miRNAs included miR-7, miR-9/miR-9*, miR-21*, miR-31/miR-31*, miR-181, miR-196a/miR-196b, miR-503 and members of the miR-29 and miR-130/301 families (Table 1). [score:3]
A set of miRNAs, miR-1, miR-18a, miR-18b, miR-19b, miR-31, miR-126, miR-142-3p, miR-133b, miR-144, miR-195, miR-223, miR-451 and miR-497 was identified with an intermediate expression level in osteosarcoma clinical samples compared to osteoblasts and bone, which may reflect the differentiation level of osteosarcoma relative to the undifferentiated osteoblast and fully differentiated normal bone. [score:2]
As predicted, the 13 miRNAs miR-1, miR-18a, miR-18b, miR-19b, miR-31, miR-126, miR-133b, miR-142-3p, miR-144, miR-195, miR-223, miR-451 and miR-497 showed opposite regulation when the osteosarcoma clinical samples were compared against bone or osteoblasts. [score:1]
These 13 miRNAs include all the above seven miRNAs (omitting miR-31*) previously described in osteoblasts [8] as well as miR-1, miR-18a, miR-18b, miR-19b, miR-133b and miR-144. [score:1]
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Inhibition of AKT or knockdown of C/EBPbeta suppressed miR-31 up-regulation. [score:9]
CUR treatment inhibited AKT activation, upregulation of C/EBPbeta and miR-31 [178]. [score:6]
EGF activation of EGFR signaling in OSCC results in the increased expression of miR-31, miR-181b and miR-222 expression. [score:5]
miR-31 is upregulated and acts as an oncogene in OSCC. [score:4]
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64
[+] score: 24
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-16-1, hsa-mir-17, hsa-mir-19a, hsa-mir-19b-1, hsa-mir-19b-2, hsa-mir-23a, hsa-mir-26a-1, hsa-mir-26b, hsa-mir-27a, hsa-mir-29a, hsa-mir-30a, hsa-mir-100, hsa-mir-29b-1, hsa-mir-29b-2, hsa-mir-16-2, mmu-mir-23b, mmu-mir-27b, mmu-mir-29b-1, mmu-mir-30a, mmu-mir-30b, mmu-mir-127, mmu-mir-128-1, mmu-mir-132, mmu-mir-133a-1, mmu-mir-188, mmu-mir-194-1, mmu-mir-195a, mmu-mir-199a-1, hsa-mir-199a-1, mmu-mir-200b, mmu-mir-205, mmu-mir-206, hsa-mir-30c-2, hsa-mir-30d, mmu-mir-122, mmu-mir-30e, hsa-mir-199a-2, hsa-mir-199b, hsa-mir-205, hsa-mir-211, hsa-mir-212, hsa-mir-214, hsa-mir-217, hsa-mir-200b, hsa-mir-23b, hsa-mir-27b, hsa-mir-30b, hsa-mir-122, hsa-mir-128-1, hsa-mir-132, hsa-mir-133a-1, hsa-mir-133a-2, hsa-mir-127, hsa-mir-138-1, hsa-mir-188, hsa-mir-194-1, hsa-mir-195, hsa-mir-206, mmu-mir-19b-2, mmu-mir-30c-1, mmu-mir-30c-2, mmu-mir-30d, mmu-mir-200a, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-16-1, mmu-mir-16-2, mmu-mir-23a, mmu-mir-26a-1, mmu-mir-26b, mmu-mir-29a, mmu-mir-29c, mmu-mir-27a, mmu-mir-31, mmu-mir-351, hsa-mir-200c, mmu-mir-17, mmu-mir-19a, mmu-mir-100, mmu-mir-200c, mmu-mir-212, mmu-mir-214, mmu-mir-26a-2, mmu-mir-211, mmu-mir-29b-2, mmu-mir-199a-2, mmu-mir-199b, mmu-mir-19b-1, mmu-mir-138-1, mmu-mir-128-2, hsa-mir-128-2, mmu-mir-217, hsa-mir-194-2, mmu-mir-194-2, hsa-mir-29c, hsa-mir-30c-1, hsa-mir-200a, hsa-mir-30e, hsa-mir-26a-2, hsa-mir-379, mmu-mir-379, mmu-mir-133a-2, mmu-mir-133b, hsa-mir-133b, mmu-mir-412, mmu-mir-431, hsa-mir-431, hsa-mir-451a, mmu-mir-451a, mmu-mir-467a-1, hsa-mir-412, hsa-mir-485, hsa-mir-487a, hsa-mir-491, hsa-mir-503, hsa-mir-504, mmu-mir-485, hsa-mir-487b, mmu-mir-487b, mmu-mir-503, hsa-mir-556, hsa-mir-584, mmu-mir-665, mmu-mir-669a-1, mmu-mir-674, mmu-mir-690, mmu-mir-669a-2, mmu-mir-669a-3, mmu-mir-669c, mmu-mir-696, mmu-mir-491, mmu-mir-504, hsa-mir-665, mmu-mir-467e, mmu-mir-669k, mmu-mir-669f, hsa-mir-664a, mmu-mir-1896, mmu-mir-1894, mmu-mir-1943, mmu-mir-1983, mmu-mir-1839, mmu-mir-3064, mmu-mir-3072, mmu-mir-467a-2, mmu-mir-669a-4, mmu-mir-669a-5, mmu-mir-467a-3, mmu-mir-669a-6, mmu-mir-467a-4, mmu-mir-669a-7, mmu-mir-467a-5, mmu-mir-467a-6, mmu-mir-669a-8, mmu-mir-669a-9, mmu-mir-467a-7, mmu-mir-467a-8, mmu-mir-669a-10, mmu-mir-467a-9, mmu-mir-669a-11, mmu-mir-467a-10, mmu-mir-669a-12, mmu-mir-3473a, hsa-mir-23c, hsa-mir-4436a, hsa-mir-4454, mmu-mir-3473b, hsa-mir-4681, hsa-mir-3064, hsa-mir-4436b-1, hsa-mir-4790, hsa-mir-4804, hsa-mir-548ap, mmu-mir-3473c, mmu-mir-5110, mmu-mir-3473d, mmu-mir-5128, hsa-mir-4436b-2, mmu-mir-195b, mmu-mir-133c, mmu-mir-30f, mmu-mir-3473e, hsa-mir-6825, hsa-mir-6888, mmu-mir-6967-1, mmu-mir-3473f, mmu-mir-3473g, mmu-mir-6967-2, mmu-mir-3473h
The study revealed downregulation of miR-205, miR-27, miR-31, and miR-29 in the cbs [+/–] retinas, these miRNAs were also reported to be downregulated in vitreous [68] and plasma of AMD patients [69]. [score:7]
In contrast, miR-31 was upregulated in the microarray data but was downregulated with validation (p value = 0.04). [score:7]
The study revealed downregulation of miR-205, miR-27, miR-31, and miR-29 in the cbs [+/–] retinas. [score:4]
Furthermore, miR-31 which was altered in cbs [+/–] retinas was suggested as a regulator of choroidal neovascularization [70]. [score:2]
miR-205, miR-27, miR-29 and miR-31 were significantly changed in our cbs [+/–] retina microarray and were also reported to be involved in AMD. [score:1]
Hcy also induces alteration of miRNAs related to tight junctions signaling such as miR-128, miR-132, miR-133, miR-195, miR-3473, miR-19, miR-200, miR-205, miR-214, miR-217, miR-23, miR-26, miR-29, miR-30, miR-31 AND miR-690. [score:1]
While intraocular injection with pre-miR-31 significantly reduced the size of the choroidal neovascular lesions. [score:1]
Other miRNAs were linked to the hypoxia signaling pathway, for instance, miR-205, miR-214, miR-217, miR-27, miR-29, miR-30 and miR-31. [score:1]
[1 to 20 of 8 sentences]
65
[+] score: 23
Divergent inverse correlation of miR-143 & HK2 expression in nonproliferative esophagus vs proliferative ZD esophageal neoplasia and human ESCCTo understand the distribution and localization of miR-143 in esophageal neoplasia in relation to localization of its target HK2 protein and the level of cell proliferation, we performed in situ hybridization (ISH) and immunohistochemical staining (IHC) on near serial sections of rat esophageal tissues (n = 10 rats/group), as well as in the archived human ESCC tissues for which we previously reported overexpression of miR-31, -21, -223 [27, 28]. [score:7]
Using the NanoString microRNA expression profiling platform, we showed that ZD promotes ESCC by inducing an oncogenic miRNA signature that resembles the human ESCC miRNAome [25] with up-regulation of oncogenic miR-31, -223, and -21 [26- 28]. [score:6]
To understand the distribution and localization of miR-143 in esophageal neoplasia in relation to localization of its target HK2 protein and the level of cell proliferation, we performed in situ hybridization (ISH) and immunohistochemical staining (IHC) on near serial sections of rat esophageal tissues (n = 10 rats/group), as well as in the archived human ESCC tissues for which we previously reported overexpression of miR-31, -21, -223 [27, 28]. [score:5]
Using the same human ESCC tissues in which we previously documented overexpression of miR-31, miR-21, miR-223 by ISH [28], Figure 4 shows these human ESCC tissues were also highly proliferative with numerous PCNA -positive nuclei (n = 12 cases). [score:3]
In situ hybridizationmiRCURY locked nucleic acid (LNA)™ microRNA detection probes, namely, hsa-miR-143, rno-miR-31, and negative controls were purchased from Exiqon (Vedbaek, Denmark). [score:1]
miRCURY locked nucleic acid (LNA)™ microRNA detection probes, namely, hsa-miR-143, rno-miR-31, and negative controls were purchased from Exiqon (Vedbaek, Denmark). [score:1]
[1 to 20 of 6 sentences]
66
[+] score: 22
Functional analyses revealed that overexpression of miR-31 considerably impaired the ability of CAFs to stimulate tumor cell migration and invasion by directly targeting the homeobox gene SATB2, which encodes a nuclear matrix-attachment protein responsible for chromatin remo deling and transcriptional regulation (reviewed in [49]). [score:7]
For instance, miR-31 inhibits expression of the forkhead box P3 (FoxP3) transcription factor that is necessary for active Treg function, providing an additional insight into a possible function for miR-31 in natural Treg -mediated suppression. [score:7]
Future work should address whether miR-31 overexpression can reduce the suppressive activity of Tregs and their effect on the immune response against tumor cells [52]. [score:5]
A good example is miR-31, which is downregulated in CAFs derived from endometrial cancer compared to paired normal endometrial fibroblasts [49]. [score:3]
[1 to 20 of 4 sentences]
67
[+] score: 22
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-15a, hsa-mir-16-1, hsa-mir-17, hsa-mir-18a, hsa-mir-19a, hsa-mir-19b-1, hsa-mir-19b-2, hsa-mir-20a, hsa-mir-21, hsa-mir-23a, hsa-mir-25, hsa-mir-26a-1, hsa-mir-27a, hsa-mir-29a, hsa-mir-30a, hsa-mir-33a, hsa-mir-92a-1, hsa-mir-92a-2, hsa-mir-93, hsa-mir-96, hsa-mir-99a, hsa-mir-100, hsa-mir-29b-1, hsa-mir-29b-2, hsa-mir-16-2, hsa-mir-198, hsa-mir-199a-1, hsa-mir-148a, hsa-mir-7-1, hsa-mir-7-2, hsa-mir-7-3, hsa-mir-10a, hsa-mir-10b, hsa-mir-34a, hsa-mir-181a-2, hsa-mir-181b-1, hsa-mir-181c, hsa-mir-182, hsa-mir-199a-2, hsa-mir-199b, hsa-mir-203a, hsa-mir-204, hsa-mir-210, hsa-mir-212, hsa-mir-181a-1, hsa-mir-214, hsa-mir-215, hsa-mir-216a, hsa-mir-217, hsa-mir-218-1, hsa-mir-218-2, hsa-mir-219a-1, hsa-mir-221, hsa-mir-222, hsa-mir-223, hsa-mir-224, hsa-let-7g, hsa-let-7i, hsa-mir-15b, hsa-mir-27b, hsa-mir-124-1, hsa-mir-124-2, hsa-mir-124-3, hsa-mir-125b-1, hsa-mir-128-1, hsa-mir-130a, hsa-mir-132, hsa-mir-135a-1, hsa-mir-135a-2, hsa-mir-142, hsa-mir-145, hsa-mir-191, hsa-mir-9-1, hsa-mir-9-2, hsa-mir-9-3, hsa-mir-125a, hsa-mir-125b-2, hsa-mir-126, hsa-mir-134, hsa-mir-146a, hsa-mir-150, hsa-mir-186, hsa-mir-188, hsa-mir-193a, hsa-mir-194-1, hsa-mir-320a, hsa-mir-155, hsa-mir-181b-2, hsa-mir-128-2, hsa-mir-194-2, hsa-mir-106b, hsa-mir-29c, hsa-mir-219a-2, hsa-mir-34b, hsa-mir-34c, hsa-mir-99b, hsa-mir-130b, hsa-mir-30e, hsa-mir-26a-2, hsa-mir-362, hsa-mir-369, hsa-mir-375, hsa-mir-378a, hsa-mir-382, hsa-mir-340, hsa-mir-328, hsa-mir-342, hsa-mir-151a, hsa-mir-148b, hsa-mir-331, hsa-mir-339, hsa-mir-335, hsa-mir-345, hsa-mir-196b, hsa-mir-424, hsa-mir-425, hsa-mir-20b, hsa-mir-451a, hsa-mir-409, hsa-mir-484, hsa-mir-486-1, hsa-mir-487a, hsa-mir-511, hsa-mir-146b, hsa-mir-496, hsa-mir-181d, hsa-mir-523, hsa-mir-518d, hsa-mir-499a, hsa-mir-501, hsa-mir-532, hsa-mir-487b, hsa-mir-551a, hsa-mir-92b, hsa-mir-572, hsa-mir-580, hsa-mir-550a-1, hsa-mir-550a-2, hsa-mir-590, hsa-mir-599, hsa-mir-612, hsa-mir-624, hsa-mir-625, hsa-mir-627, hsa-mir-629, hsa-mir-33b, hsa-mir-633, hsa-mir-638, hsa-mir-644a, hsa-mir-650, hsa-mir-548d-1, hsa-mir-449b, hsa-mir-550a-3, hsa-mir-151b, hsa-mir-320b-1, hsa-mir-320c-1, hsa-mir-454, hsa-mir-320b-2, hsa-mir-378d-2, hsa-mir-708, hsa-mir-216b, hsa-mir-1290, hsa-mir-320d-1, hsa-mir-320c-2, hsa-mir-320d-2, hsa-mir-378b, hsa-mir-3151, hsa-mir-320e, hsa-mir-378c, hsa-mir-550b-1, hsa-mir-550b-2, hsa-mir-378d-1, hsa-mir-378e, hsa-mir-378f, hsa-mir-378g, hsa-mir-378h, hsa-mir-378i, hsa-mir-219b, hsa-mir-203b, hsa-mir-451b, hsa-mir-499b, hsa-mir-378j, hsa-mir-486-2
miR-31 was down-regulated in ATL via epigenetic regulation and it caused up-regulation of its target gene, NIK, which activated the NF-κB signaling pathway and caused apoptosis resistance [148]. [score:10]
Several studies have reported deregulated microRNAs in ATL patient samples and HTLV-1-transformed cells, among them miR-155, miR-146a, miR-150, and miR-223 were found up-regulated and miR-31 and miR124a down-regulated [146– 149]. [score:8]
Deregulation of miR-146a, miR-155, miR-150 and miR-223 was reported to affect cellular proliferation [151– 153] and alteration of miR-31, miR-130b and miR-93 were involved in apoptosis resistance [154], suggesting a possible role of miRNA expression in ATL progression and pathogenesis. [score:4]
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68
[+] score: 22
Neither miR-24 nor miR-31 alone was sufficient to alter development of multipotent MSCs to mature adipocytes, but in the presence of BMP2, miR-24 overexpression accelerated mature adipocyte marker expression, while miR-31 overexpression suppressed the adipogenic markers PPARG, CEBPA and aP2 [64]. [score:10]
Therefore, miR-31 downregulation could indirectly suppress leptin via translational repression of CEBPA during adipogenic lineage commitment of MSCs [64]. [score:9]
A putative miR-31 binding site in the 3’UTR of CEBPA was experimentally validated [64]. [score:1]
Unlike mouse MSCs miR-31 and miR-24 were not reported to be altered during adipogenic differentiation of human multipotent MSCs [75, 76]. [score:1]
Opposing effects of miR-24 and miR-31 have been reported in the C3H10T1/2 multipotent mouse embryonic stem cell line treated with BMP2 to induce adipogenic differentiation [64]. [score:1]
[1 to 20 of 5 sentences]
69
[+] score: 22
Mitra et al. found that, in ovarian CAFs, miR-31 and miR-214 are downregulated while miR-155 is upregulated compared to normal or tumor-adjacent fibroblasts [87]. [score:6]
They discovered that miR-31, the least regulated miR in serous ovarian cancer, repressed the cell cycle regulator E2F2, inhibited proliferation, and induced apoptosis. [score:5]
Lower expression of miR-31 and higher expression of MET (also known as c-Met or hepatocyte growth factor receptor) were significantly correlated with PTX resistance and poor prognosis in ovarian cancer patients. [score:5]
miR-31 directly targets the 3′-UTR of MET and increases the PTX sensitivity of ovarian cancer cells in an animal mo del. [score:4]
They revealed that loss of miR-31 is associated with defects in the TP53 (also called p53) pathway and functions in serous ovarian cancer, suggesting that patients with cancers that are deficient in TP53 activity might benefit from therapeutic delivery of miR-31. [score:1]
6.6. miR-31. [score:1]
[1 to 20 of 6 sentences]
70
[+] score: 21
MiR-31 has been shown to act as an oncogenic miRNA by targeting specific tumor suppressors, including the large tumor suppressor 2 (LATS2) and PP2A regulatory subunit B alpha isoform (PPP2R2A) [39]. [score:8]
A meta-analysis in various cancers has shown the overexpression of miR-31 [38], which was overexpressed in early stages, and expression was high in tumor progression and reached higher levels in advanced stages. [score:7]
Regarding the analyzed miRNAs, the overexpression of miR-182, miR-31, miR135b, miR-199b, miR-224 and miR-196b and miR-34a have been detected in both the training and validation set. [score:3]
An analysis of the predicted targets of miR-31 found a relationship between this miRNAs and the initiation, progression and treatment response of lung cancer through the cell cycle, the cytochrome P450 pathway, metabolic pathways, apoptosis, the chemokine signaling pathway, and the MAPK signaling pathway [40]. [score:3]
[1 to 20 of 4 sentences]
71
[+] score: 21
miR-31, a reported target of hif1a, was increased in specimens obtained from patients with active ulcerative colitis [31] and during progression and neoplastic transformation of IBD, [32] but it was down-regulated in NEC tissues (0.12 fold). [score:6]
When miRNAs such as miR-31 and miR-203 were down-regulated in NEC tissues, they could potentiate the upsurge of proinflammatory signals mediated by TLR4, key transcription factor NFκB, hypoxia/oxidative regulators HIF1A and PTGS2, inflammatory cytokines and chemokines TNF and IL8, as well as angiogenic factor HBEGF, resulting in extensive mucosal injury (Fig 3). [score:5]
Although many IBD -associated miRNAs did not appear to be dysregulated in preterm NEC tissues, several miRNAs, including miR-223, miR-132, miR-146b-3p, miR-215, miR-375, miR-31 and miR-141, were regulated in both IBD and NEC. [score:3]
Some dysregulated miRNAs such as miR-1290, miR-146b-3p, miR-31, miR-375 and miR-200a have established GI functions (S3 Table). [score:2]
A proposed network of dysregulated miRNA/mRNA pairs in NEC suggested interaction at bacterial receptor TLR4 (miR-31, miR-451, miR-203, miR-4793-3p), mediated via key transcription factors NFKB2 (miR-203), AP-1 /FOSL1 (miR-194-3p), FOXA1 (miR-21-3p, miR-431 and miR-1290) and HIF1A (miR-31), and extended downstream to pathways of angiogenesis, arginine metabolism, cell adhesion and chemotaxis, extracellular matrix remo deling, hypoxia/oxidative stress, inflammation and muscle contraction. [score:2]
If proven effective at multiple hierarchies of the inflammatory cascade, specific miRNAs such as miR-31, miR-203 or miR-194-3p, could be developed for treatment to dampen the exaggerated immunologic response that contributes to the pathophysiology of NEC. [score:1]
Interestingly, we identified miRNAs that could exhibit multiple interacting points at the upstream receptor and transcription factors as well as downstream effector genes e. g., miR-31 (TLR4, HIF1A, HBEGF), miR-194-3p (AP-1 /FOSL1, MMP9, TIMP1), miR-203 (TLR4, NFKB2, HBEGF, IL8, TNF, PTGS2), miR-1290 (FOXA1, THBS1) and miR-200b-5p (SOD2, FLT1). [score:1]
Levels of 15 miRNAs: miR-223, miR-1290, miR-4725-3p, miR-4793-3p, miR-410, miR-187, miR-375, miR-203, miR-200b-5p, miR-194-3p, miR-200a, miR-215, miR-31, miR-192-3p and miR-141 were significantly different between NEC and SIP tissues (0.12–59.05 fold; Table 2). [score:1]
[1 to 20 of 8 sentences]
72
[+] score: 21
As discussed above, it was also shown that the oncogenic NOTCH1/MYC pathway inhibited the expression of tumor suppressor miRNAs, including miR-31, miR-150, and miR-155 [43]. [score:7]
Interestingly, it was also noted that the oncogenic NOTCH1/MYC pathway inhibited miR-31, miR-150, and miR-155 expression. [score:5]
Enforced expression of a set of tumor suppressor miRNAs (miR-29, miR-31, miR-150, miR-155, and miR-200) had anti-proliferative effects in four human T-ALL cell lines [43]. [score:5]
They also discovered a potential new oncogene in T-ALL, HBP1 (target of miR-29, miR-31, miR-155, and miR-200), which encodes a transcription factor. [score:3]
After selection, five miRNAs were characterized (miR-29, miR-31, miR-150, miR-155, and miR-200), all with tumor-suppressive effects in NOTCH1 -driven T-ALL mo del. [score:1]
[1 to 20 of 5 sentences]
73
[+] score: 21
Other miRNAs from this paper: hsa-mir-331
Furthermore, to detect the transfection efficiency and eliminate the influence of miR-31 may exist on overexpression of Loc554202, the expression levels of Loc554202 and miR-31 were detected by a qRT-PCR analysis after transfecting these cells with pCDNA-Loc554202. [score:5]
The expression level of miR-31 was no significant change after transfecting CRC cells with pCDNA-Loc554202, which eliminated the influence of miR-31 may exist on overexpression of Loc554202. [score:5]
The results found that the Loc554202 expression was increased by 47.5-fold and 38-fold in the HCT116 and DLD1 cells, respectively, compared with the control cells following transfection with pCDNA-Loc554202 (Fig.   1e), and there was no significant change in miR-31 expression (Additional file 3: Figure S1). [score:4]
The relative expression levels of miR-31 following the treatment of HCT116 and DLD1 cells with pCDNA-Loc554202 and empty vector. [score:3]
A previous study demonstrated that Loc554202 as the host gene of miR-31 regulates the proliferation and migration of breast cancer cells [22], and CpG island methylation plays an important role in silencing the Loc554202 genes [23]. [score:2]
Loc554202, is a 2166-bp transcript on human chromosome 9p21.3, which is the host gene of miR-31 and dysregulated in breast [22, 23] and lung [24] cancer cells, although the importance of its function has not yet been established. [score:2]
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74
[+] score: 20
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-17, hsa-mir-19a, hsa-mir-21, hsa-mir-92a-1, hsa-mir-92a-2, hsa-mir-105-1, hsa-mir-105-2, hsa-mir-199a-1, hsa-mir-34a, hsa-mir-187, hsa-mir-199a-2, hsa-mir-205, hsa-mir-214, hsa-mir-221, hsa-let-7g, hsa-let-7i, hsa-mir-128-1, hsa-mir-141, hsa-mir-144, hsa-mir-145, hsa-mir-9-1, hsa-mir-9-2, hsa-mir-9-3, hsa-mir-146a, hsa-mir-200c, hsa-mir-128-2, hsa-mir-29c, hsa-mir-376a-1, hsa-mir-378a, hsa-mir-133b, hsa-mir-429, hsa-mir-487a, hsa-mir-515-1, hsa-mir-515-2, hsa-mir-526b, hsa-mir-514a-1, hsa-mir-514a-2, hsa-mir-514a-3, hsa-mir-376a-2, hsa-mir-548a-1, hsa-mir-548b, hsa-mir-548a-2, hsa-mir-548a-3, hsa-mir-548c, hsa-mir-548d-1, hsa-mir-548d-2, hsa-mir-656, hsa-mir-542, hsa-mir-378d-2, hsa-mir-548e, hsa-mir-548j, hsa-mir-548k, hsa-mir-548l, hsa-mir-548f-1, hsa-mir-548f-2, hsa-mir-548f-3, hsa-mir-548f-4, hsa-mir-548f-5, hsa-mir-548g, hsa-mir-548n, hsa-mir-548m, hsa-mir-548o, hsa-mir-548h-1, hsa-mir-548h-2, hsa-mir-548h-3, hsa-mir-548h-4, hsa-mir-1275, hsa-mir-548p, hsa-mir-548i-1, hsa-mir-548i-2, hsa-mir-548i-3, hsa-mir-548i-4, hsa-mir-2114, hsa-mir-548q, hsa-mir-548s, hsa-mir-378b, hsa-mir-548t, hsa-mir-548u, hsa-mir-548v, hsa-mir-548w, hsa-mir-548x, hsa-mir-514b, hsa-mir-378c, hsa-mir-4303, hsa-mir-4309, hsa-mir-4307, hsa-mir-4278, hsa-mir-548y, hsa-mir-548z, hsa-mir-548aa-1, hsa-mir-548aa-2, hsa-mir-548o-2, hsa-mir-378d-1, hsa-mir-378e, hsa-mir-548h-5, hsa-mir-548ab, hsa-mir-378f, hsa-mir-378g, hsa-mir-548ac, hsa-mir-548ad, hsa-mir-548ae-1, hsa-mir-548ae-2, hsa-mir-548ag-1, hsa-mir-548ag-2, hsa-mir-548ah, hsa-mir-378h, hsa-mir-548ai, hsa-mir-548aj-1, hsa-mir-548aj-2, hsa-mir-548x-2, hsa-mir-548ak, hsa-mir-548al, hsa-mir-378i, hsa-mir-548am, hsa-mir-548an, hsa-mir-548ao, hsa-mir-548ap, hsa-mir-548aq, hsa-mir-548ar, hsa-mir-548as, hsa-mir-548at, hsa-mir-548au, hsa-mir-548av, hsa-mir-548aw, hsa-mir-548ax, hsa-mir-378j, hsa-mir-548ay, hsa-mir-548az, hsa-mir-548ba, hsa-mir-548bb, hsa-mir-548bc
Twenty-six among 30 downregulated genes have been reported before such as hsa-miR-31, hsa-miR-1275, hsa-miR-526b, hsa-miR-2114, and hsa-miR-378c; and hsa-miR-4303, hsv2-miR-H13, and hsv2-miR-H10 were first reported with downregulation in gastric cancer. [score:7]
For example, in breast cancer loss of has-miR-31 expression is associated with high risk metastases [22], whereas in colorectal cancer high has-miR-31 expression correlates with advanced disease stage [23]. [score:7]
In the present study, the miRNA chip revealed that the has-miR-31 was downregulated in gastric cancer tissues, consistent with a previous study by Zhang et al., which had identified that has-miR-31 was lower in cancer tissues in comparison with noncancerous tissues. [score:4]
has-miR-31 was frequently altered in a large variety of cancers. [score:1]
Among them hsa-miR-31 and has-miR-133b were included which were screened in our findings [21]. [score:1]
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75
[+] score: 19
Mature miRNA expression could be classified into two groups: i) cardia-tissues: miRNAs rarely expressed in other tissues but expressed in gastric cardia, including miR-148a, miR-192, miR-200a and miR-200b; ii) quasi-ubiquitous: miRNAs expressed in many tissues and conditions, including miR-29c, miR-21, miR-24, miR-29b, miR-29a, miR-451, miR-31, miR-145, miR-26a, miR-19b and let-7b. [score:9]
Six miRNAs showed a low variable pattern of expression (miR-29b, miR-29c, miR-19b, miR-31, miR-148a, miR-451) and could be considered part of the expression pattern of the healthy gastric tissue. [score:4]
Could observe miRNAs with high interindividual variation, for exempla miR-21, and another with low interindividual variation, e. g. expression pattern slightly variable (miR-29b, miR-29c, miR-19b, miR-31, miR-148a, miR-451). [score:3]
The high expression levels of miRNAs identified by ultra-deep sequencing (in descending order: miR-29c, miR-21, miR-148a, miR-29a, miR-24, miR-29b, miR-192, miR-451, miR-145, miR-31, miR-200a, miR-19b, miR-200b, let-7b and miR-26a) were validated with the TaqMan miRNA assays (Life Technologies). [score:2]
hsa-miR-31 ANKRD52 ; TNRC6B ; NFAT5 ; BACH2 ; NUFIP2 ; KIAA2018 ; SLC16A2 ; PLXNA4 ; C11orf41 ; DICER1 ; SNTB2 ; ELAVL2; CCNJ; CALCR; BAHD1; RHOBTB1; ZBTB34; RAPH1; SLC1A2; JAZF1. [score:1]
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[+] score: 19
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-15a, hsa-mir-16-1, hsa-mir-17, hsa-mir-18a, hsa-mir-21, hsa-mir-24-1, hsa-mir-24-2, hsa-mir-25, hsa-mir-27a, hsa-mir-30a, hsa-mir-29b-1, hsa-mir-29b-2, hsa-mir-103a-2, hsa-mir-103a-1, hsa-mir-16-2, mmu-let-7g, mmu-let-7i, mmu-mir-15b, mmu-mir-29b-1, mmu-mir-30a, mmu-mir-30b, mmu-mir-125a, mmu-mir-125b-2, mmu-mir-126a, mmu-mir-127, mmu-mir-9-2, mmu-mir-141, mmu-mir-145a, mmu-mir-155, mmu-mir-10b, mmu-mir-24-1, mmu-mir-205, mmu-mir-206, hsa-mir-30c-2, hsa-mir-30d, mmu-mir-30e, hsa-mir-10b, hsa-mir-34a, hsa-mir-205, hsa-mir-221, mmu-mir-290a, mmu-mir-34c, mmu-mir-34b, mmu-let-7d, mmu-mir-106b, hsa-let-7g, hsa-let-7i, hsa-mir-15b, hsa-mir-30b, hsa-mir-125b-1, hsa-mir-141, hsa-mir-145, hsa-mir-9-1, hsa-mir-9-2, hsa-mir-9-3, hsa-mir-125a, hsa-mir-125b-2, hsa-mir-126, hsa-mir-127, hsa-mir-206, mmu-mir-30c-1, mmu-mir-30c-2, mmu-mir-30d, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7b, mmu-let-7c-1, mmu-let-7c-2, mmu-let-7e, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-15a, mmu-mir-16-1, mmu-mir-16-2, mmu-mir-18a, mmu-mir-21a, mmu-mir-24-2, mmu-mir-27a, mmu-mir-31, mmu-mir-34a, mmu-mir-103-1, mmu-mir-103-2, mmu-mir-322, hsa-mir-200c, hsa-mir-155, mmu-mir-17, mmu-mir-25, mmu-mir-200c, mmu-mir-221, mmu-mir-29b-2, mmu-mir-9-1, mmu-mir-9-3, mmu-mir-125b-1, hsa-mir-106b, hsa-mir-30c-1, hsa-mir-34b, hsa-mir-34c, hsa-mir-30e, hsa-mir-373, hsa-mir-20b, hsa-mir-520c, hsa-mir-503, mmu-mir-20b, mmu-mir-503, hsa-mir-103b-1, hsa-mir-103b-2, mmu-mir-145b, mmu-mir-21b, mmu-let-7j, mmu-mir-21c, mmu-mir-30f, mmu-let-7k, mmu-mir-126b, mmu-mir-290b, mmu-mir-9b-2, mmu-mir-9b-1, mmu-mir-9b-3
The miR-31, one of the highly cited tumor suppressor miRNAs in human breast cancer, was also found to be down-regulated and differentially expressed in canine osteosarcoma and mammary tumors, respectively [80]. [score:8]
The overexpression of certain oncogenic miRNAs (miR-21, miR-27a, miR-155, miR-9, miR-10b, miR-373/miR-520c, miR-206, miR-18a/b, miR-221/222) and the loss of several tumor suppressor miRNAs (miR-205/200, miR-125a, miR-125b, miR-126, miR-17-5p, miR-145, miR-200c, let-7, miR-20b, miR-34a, miR-31, miR-30) lead to loss of regulation of vital cellular functions that are involved in breast cancer pathogenesis [127, 128]. [score:6]
Expression of p16/INK4A is repressed by miR-24 and miR-31 which are also involved in the regulation of cell proliferation and progression of cell cycle in many cancers [141, 142]. [score:4]
Furthermore, the region at canine chromosome 11 (orthologous to human chromosome 9p21) encoding INK4A/ARF, MTAP and close neighbors including miR-31, as shown in the comparative chromosomal mapping (Figure 4), is also highly susceptible and prone to concomitant deletion in many cancers in dogs [16]. [score:1]
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[+] score: 19
An increase of miR-30c and miR-31 miRNAs, targeting osteogenic transcripts such as RUNX2 and Osterix, [23, 26- 28] and of miR-125a known to be significantly downregulated during osteogenic differentiation in human adipose-derived stem cells [38] and predicted to target the osteogenic genes Smad 2 and 4 [39], could be found in hOst incubated with hAdi-CM. [score:8]
Five miRNAs, miR-138, miR-30c, miR-125a, miR-125b and miR-31, were selected for their capacity to inhibit osteoblast gene expression [25- 28]. [score:5]
We observed in the osteoblastic population an increase in the adipogenic PPARγ, leptin, CEBPα and CEBPδ transcripts, dependent on mRNA amount as shown by conditioned media obtained from adipocytes at several differentiation stages and PPARγ silencing experiments, as well as the anti-osteoblastic miR-138, miR30c, miR125a, miR-125b, miR-31 miRNAs [23- 26], probably implicated in osteocalcin (OC) and osteopontin (OP) expression decrease. [score:3]
We observed in hMSC-Ost incubated in hAdi-CM an increase in the adipogenic PPARγ, leptin, CEBPα and CEBPδ transcripts as well as the anti-osteoblastic miR-138, miR30c, miR125a, miR-125b, miR-31 miRNAs, probably implicated in the observed osteocalcin (OC) and osteopontin (OP) expression decrease. [score:3]
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[+] score: 18
Five of these [miR-34b-3p, miR-34c-5p, miR-34b-5p, miR-92b-3p, and miR-182-5p; as well as miR-31-5p, which was identified through literature search (41)] belonged to the aforementioned seven miRNAs which were expressed more highly in the C57BL/6J mice and downregulated throughout the time course. [score:6]
Furthermore, seven miRNAs were expressed more highly in C57BL/6J mice and were mainly downregulated across the time course (miR-92b-3p, miR-34b-5p, miR-672-5p, miR-31-5p, miR-34c-5p, miR-34b-3p, and miR-182-5p; listed in descending order according to the heat map in Figure 5). [score:6]
Higher abundance of antiapoptotic (e. g., miR-467 family) and lower abundance of proapoptotic miRNAs (e. g., miR-34 family) and those regulating the PI3K-Akt pathway (e. g., miR-31-5p) were associated with the more susceptible DBA/2J strain. [score:2]
Two of the 20 miRNAs (miR-31-5p and miR-182-5p) can be linked to adaptive immunity, i. e., T cell activation and regulation of Treg differentiation, respectively (72, 73). [score:2]
Using the ViTa Database, the human homologs of miR-135b-5p, miR-147-3p, miR-31-5p, miR-379-5p, miR-7a-5p, as well as the miR-449 (-5p) and miR-34 (-5p) families, were predicted to bind to viral RNA segments of influenza A/Puerto Rico/8/34/Mount Sinai (H1N1). [score:1]
Of note, miR-31-5p, miR-379-5p, miR-7a-5p, as well as some members of the miR-449 (-5p) and miR-34 (-5p) families were moderately to highly abundant (>10 CPM), making it more likely that they would bind to a biologically relevant number of viral RNAs. [score:1]
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Further study confirmed this result showing that miR-31 expression was positively related to advanced TNM stage and deeper invasion of tumors, suggesting over -expression of miR-31 might be involved in the development and progression of CRC [104]. [score:6]
Another study found that miR-21 and miR-31 were significantly up-regulated, and miR-143 and miR-145 down-regulated in tumors compared with the normal counterparts. [score:6]
The expression level of miR-31 was correlated with the stage of CRC [59]. [score:3]
Moreover, the expression levels of miR-31 were higher in the tumor samples and CRC cell lines in comparison to the non-tumoral samples and were related to pathological stage, suggesting that this miRNA could contribute to both, the tumorogenesis and the acquisition of a more aggressive phenotype in CRC. [score:3]
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[+] score: 18
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-16-1, hsa-mir-17, hsa-mir-21, hsa-mir-23a, hsa-mir-24-1, hsa-mir-24-2, hsa-mir-25, hsa-mir-26a-1, hsa-mir-26b, hsa-mir-30a, hsa-mir-96, hsa-mir-99a, hsa-mir-16-2, hsa-mir-30c-2, hsa-mir-30d, hsa-mir-182, hsa-mir-183, hsa-mir-211, hsa-mir-217, hsa-mir-218-1, hsa-mir-218-2, hsa-mir-221, hsa-mir-222, hsa-let-7g, hsa-let-7i, hsa-mir-15b, hsa-mir-23b, hsa-mir-30b, hsa-mir-125b-1, hsa-mir-132, hsa-mir-143, hsa-mir-145, hsa-mir-191, hsa-mir-125a, hsa-mir-125b-2, hsa-mir-126, hsa-mir-184, hsa-mir-190a, hsa-mir-195, rno-mir-322-1, rno-let-7d, rno-mir-335, rno-mir-342, rno-mir-135b, hsa-mir-30c-1, hsa-mir-299, hsa-mir-30e, hsa-mir-26a-2, hsa-mir-379, hsa-mir-382, hsa-mir-342, hsa-mir-135b, hsa-mir-335, rno-let-7a-1, rno-let-7a-2, rno-let-7b, rno-let-7c-1, rno-let-7c-2, rno-let-7e, rno-let-7f-1, rno-let-7f-2, rno-let-7i, rno-mir-15b, rno-mir-16, rno-mir-17-1, rno-mir-21, rno-mir-23a, rno-mir-23b, rno-mir-24-1, rno-mir-24-2, rno-mir-25, rno-mir-26a, rno-mir-26b, rno-mir-30c-1, rno-mir-30e, rno-mir-30b, rno-mir-30d, rno-mir-30a, rno-mir-30c-2, rno-mir-31a, rno-mir-96, rno-mir-99a, rno-mir-125a, rno-mir-125b-1, rno-mir-125b-2, rno-mir-126a, rno-mir-132, rno-mir-143, rno-mir-145, rno-mir-183, rno-mir-184, rno-mir-190a-1, rno-mir-191a, rno-mir-195, rno-mir-211, rno-mir-217, rno-mir-218a-2, rno-mir-218a-1, rno-mir-221, rno-mir-222, rno-mir-299a, hsa-mir-384, hsa-mir-20b, hsa-mir-409, hsa-mir-412, hsa-mir-489, hsa-mir-494, rno-mir-489, rno-mir-412, rno-mir-543, rno-mir-542-1, rno-mir-379, rno-mir-494, rno-mir-382, rno-mir-409a, rno-mir-20b, hsa-mir-542, hsa-mir-770, hsa-mir-190b, hsa-mir-543, rno-mir-466c, rno-mir-17-2, rno-mir-182, rno-mir-190b, rno-mir-384, rno-mir-673, rno-mir-674, rno-mir-770, rno-mir-31b, rno-mir-191b, rno-mir-299b, rno-mir-218b, rno-mir-126b, rno-mir-409b, rno-let-7g, rno-mir-190a-2, rno-mir-322-2, rno-mir-542-2, rno-mir-542-3
MiRNAs found to be primarily down-regulated in DHT -treated rats includes rno-miR-770, rno-miR-466c, rno-miR-21, rno-miR-31, rno-miR-182, rno-miR-183, rno-miR-96, rno-miR-132, rno-miR-182, rno-miR-384-3p and rno-miR-184. [score:4]
Thus, it is possible that the down-regulation of miRNAs (rno-miR-770, rno-miR-466c, rno-miR-31, rno-miR-183, rno-miR-96, rno-miR-132, rno-miR-182, rno-miR-384-3p and rno-miR-184) observed in this study could be associated with promoted thecal hyperandrogenesis [37, 38]. [score:4]
Whereas rno-miR-24 and rno-miR-183 were highly expressed in the theca and, to a lesser extent, in the granulosa cells of the cystic follicles (Figure  5), Rno-miR-31 and rno-miR-96 were present in the cumulus granulosa cells. [score:3]
Among the fourteen miRNAs mapped to the ingenuity databases, twelve (rno-let-7d, rno-miR-132, rno-miR-182, rno-miR-183, rno-miR-184, rno-miR-21, rno-miR-221, rno-miR-24, rno-miR-25, rno-miR-26b, rno-miR-31 and rno-miR-96) had 171 experimentally validated targets. [score:3]
For example, rno-miR-96, rno-miR-31 and rno-miR-222 were exclusively expressed in the theca of cystic follicles. [score:3]
These included rno-miR-24, rno-miR-31, rno-miR-96, rno-miR-183, rno-miR-222, rno-miR-489, U6 snRNA (positive control) and scrambled miRNA (negative control). [score:1]
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[+] score: 18
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-17, hsa-mir-18a, hsa-mir-19b-1, hsa-mir-19b-2, hsa-mir-21, hsa-mir-23a, hsa-mir-92a-1, hsa-mir-92a-2, hsa-mir-96, hsa-mir-98, hsa-mir-99a, hsa-mir-106a, mmu-let-7g, mmu-let-7i, mmu-mir-23b, mmu-mir-99a, mmu-mir-127, mmu-mir-128-1, mmu-mir-136, mmu-mir-142a, mmu-mir-145a, mmu-mir-10b, mmu-mir-182, mmu-mir-183, mmu-mir-187, mmu-mir-193a, mmu-mir-195a, mmu-mir-200b, mmu-mir-206, mmu-mir-143, hsa-mir-139, hsa-mir-10b, hsa-mir-182, hsa-mir-183, hsa-mir-187, hsa-mir-210, hsa-mir-216a, hsa-mir-217, hsa-mir-219a-1, hsa-mir-221, hsa-mir-222, hsa-mir-224, hsa-mir-200b, mmu-mir-302a, mmu-let-7d, mmu-mir-106a, hsa-let-7g, hsa-let-7i, hsa-mir-23b, hsa-mir-128-1, hsa-mir-142, hsa-mir-143, hsa-mir-145, hsa-mir-127, hsa-mir-136, hsa-mir-193a, hsa-mir-195, hsa-mir-206, mmu-mir-19b-2, mmu-mir-200a, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7b, mmu-let-7c-1, mmu-let-7c-2, mmu-let-7e, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-18a, mmu-mir-21a, mmu-mir-23a, mmu-mir-31, mmu-mir-92a-2, mmu-mir-96, mmu-mir-98, hsa-mir-200c, mmu-mir-17, mmu-mir-139, mmu-mir-200c, mmu-mir-210, mmu-mir-216a, mmu-mir-219a-1, mmu-mir-221, mmu-mir-222, mmu-mir-224, mmu-mir-19b-1, mmu-mir-92a-1, mmu-mir-128-2, hsa-mir-128-2, mmu-mir-217, hsa-mir-200a, hsa-mir-302a, hsa-mir-219a-2, mmu-mir-219a-2, hsa-mir-363, mmu-mir-363, hsa-mir-302b, hsa-mir-302c, hsa-mir-302d, hsa-mir-371a, hsa-mir-18b, hsa-mir-20b, hsa-mir-452, mmu-mir-452, ssc-mir-106a, ssc-mir-145, ssc-mir-216-1, ssc-mir-217-1, ssc-mir-224, ssc-mir-23a, ssc-mir-183, ssc-let-7c, ssc-let-7f-1, ssc-let-7i, ssc-mir-128-1, ssc-mir-136, ssc-mir-139, ssc-mir-18a, ssc-mir-21, hsa-mir-146b, hsa-mir-493, hsa-mir-495, hsa-mir-497, hsa-mir-505, mmu-mir-20b, hsa-mir-92b, mmu-mir-302b, mmu-mir-302c, mmu-mir-302d, hsa-mir-671, mmu-mir-216b, mmu-mir-671, mmu-mir-497a, mmu-mir-495, mmu-mir-146b, mmu-mir-708, mmu-mir-505, mmu-mir-18b, mmu-mir-493, mmu-mir-92b, hsa-mir-708, hsa-mir-216b, hsa-mir-935, hsa-mir-302e, hsa-mir-302f, ssc-mir-17, ssc-mir-210, ssc-mir-221, mmu-mir-1839, ssc-mir-146b, ssc-mir-206, ssc-let-7a-1, ssc-let-7e, ssc-let-7g, ssc-mir-128-2, ssc-mir-143, ssc-mir-10b, ssc-mir-23b, ssc-mir-193a, ssc-mir-99a, ssc-mir-98, ssc-mir-92a-2, ssc-mir-92a-1, ssc-mir-92b, ssc-mir-142, ssc-mir-497, ssc-mir-195, ssc-mir-127, ssc-mir-222, ssc-mir-708, ssc-mir-935, ssc-mir-19b-2, ssc-mir-19b-1, ssc-mir-1839, ssc-mir-505, ssc-mir-363-1, hsa-mir-219b, hsa-mir-371b, ssc-let-7a-2, ssc-mir-18b, ssc-mir-187, ssc-mir-218b, ssc-mir-219a, mmu-mir-195b, mmu-mir-145b, mmu-mir-21b, mmu-let-7j, mmu-mir-21c, ssc-let-7d, ssc-let-7f-2, ssc-mir-20b-1, ssc-mir-20b-2, ssc-mir-31, ssc-mir-182, ssc-mir-216-2, ssc-mir-217-2, ssc-mir-363-2, ssc-mir-452, ssc-mir-493, ssc-mir-671, mmu-let-7k, ssc-mir-7138, mmu-mir-219b, mmu-mir-216c, mmu-mir-142b, mmu-mir-497b, mmu-mir-935, ssc-mir-9843, ssc-mir-371, ssc-mir-219b, ssc-mir-96, ssc-mir-200b
Compared with pEFs, expression of ssc-miR-145-5p and ssc-miR-98 were down-regulated in both two types of piPSCs (Fig 2D), whereas ssc-miR-1839-5p and ssc-miR-31 were both up-regulated in both types of piPSCs (Fig 2E). [score:8]
To validate the differential expression identified by the miRNA sequencing, ssc-miR-145-5p, ssc-miR-98, ssc-miR-31 and ssc-miR-1839-5p were selected for quantitative stem-loop RT-PCR analysis. [score:3]
The relative expression of ssc-miR-31 and ssc-miR-1839-5p performed by quantitative stem-loop RT-PCR was consistent with small RNA sequencing. [score:3]
Ssc-miR-31 was highly expressed in mESCs, as well as in somatic stem cells [40, 41]. [score:3]
Among these DE miRNAs, ssc-miR-31 and ssc-miR-1839-5p were selected to confirm the small RNA sequencing results. [score:1]
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