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135 publications mentioning mmu-mir-7a-2 (showing top 100)

Open access articles that are associated with the species Mus musculus and mention the gene name mir-7a-2. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1
[+] score: 448
Other miRNAs from this paper: mmu-mir-7a-1, mmu-mir-7b
FADD knockdown was associated with down-regulation of FAK but up-regulation of miR-7a (Figure 4C, 4D, 4G and 4H) while FADD overexpression was associated with up-regulation of FAK but down-regulation of miR-7a (Figure 4E, 4F, 4I and 4J), consistent with the results from MEF cells. [score:16]
Since the basic FAK expression level in B16F10 was originally high, we chose B16F1 to study FAK expression and cell migratory ability in cells with up and down-regulated miR-7a along with FADD overexpression (Figure 7B, 7H, 7I and 7J). [score:10]
Similarly, up- or down-regulated miR-7a expression with mimic or inhibitor would have the homoplastic effect on FADD -overexpressed cells. [score:10]
As shown in Figure 7G, B16F10 cells treated with FADD siRNA scramble (NC) plus miR-7a inhibitor exhibited higher protein level of FAK than NC plus miR-7a inhibitor nc, but the protein level of FAK in B16F10 cells treated with siFADD plus miR-7a inhibitor significantly decreased, though it did not decrease as much as that in B16F10 cells treated with its control DNA (siFADD plus miR-7a inhibitor nc). [score:9]
B. and C. When FADD was knockdown by RNAi (B) or overexpressed by transfected pRK5-FADD plasmid (C), the expression of miR-7a reversely correlated to FADD expression in MEFs. [score:8]
To validate whether FAK is a miR-7a target gene in murine melanoma cells, FAK mRNA and protein expression was determined in B16F10 cells after knockdown or overexpression of miR-7a. [score:8]
Conversely, FAK protein expression was up-regulated in B16F10 cells transfected with miR-7a inhibitor. [score:8]
Interestingly, we also observed suppression of FAK expression which retarded cell migration caused by FADD interference can be abrogated by recovering miR-7a expression level. [score:7]
Several groups have reported that miR-7 inhibits migration and invasion via targeting FAK expression in human breast, cervical and glioblastoma cancer cells [28– 30]. [score:7]
According to our hypothesis, if the weakening of FAK -induced migration by FADD inhibition was mediated by miR-7a, miR-7a inhibitor would rescue cells from retarded migration whereas miR-7a mimic would further inhibit the migratory ability. [score:7]
As the systematic schema showed in Figure 7K, by elevating the expression of miR-7a, FADD interference could decrease the expression of FAK expression. [score:7]
All synthetic miRNA products including miR-7a mimic (dsRNA oligonucleotides), negative control mimic (NC) (micrON™ mimic Negative Control #22, Standard, 2nmol), miR-7a inhibitors, inhibitor negative control (micrOFF™ inhibitor Negative Control #22, Standard, 2nmol) were purchased from RiBoBio (Guangzhou, China). [score:7]
Although we have not elucidated a detailed mechanism of how FADD regulates the expression of miR-7a, we suspect that FADD may regulate miR-7a's expression by altering transcriptional activity of hnRNP K pre-mRNA transcript from which miR-7a is derived [40]. [score:7]
To show that miR-7a inhibits the expression of the murine FAK gene by directly binding to its 3′UTR, the murine FAK 3′UTR was amplified and cloned into the pmirGLO Vector (Promega, USA), downstream of the luciferase gene, named pGL-FAK-3′UTR. [score:6]
Expression of miR-7a was up-regulated in FADD−/− MEFs. [score:6]
Microarray analysis revealed an up-regulation of miR-7a expression in FADD [−/−] MEFs. [score:6]
Likewise, Figure 7J showed that the up or down-regulation of miR-7a could decrease or increase the expression of FADD -induced FAK in B16F1 cells, respectively. [score:6]
However, when miR-7a was directly inhibited by an inhibitor a much more prominent change in FAK can be observed in B16F10 (Figure 5B). [score:6]
Expression of miR-7a was up-regulated in FADD -deficient MEFs. [score:6]
Inhibiting miR-7a expression rescued FADD interference reduced cell migration. [score:5]
With the help of microarray and qRT-PCR analysis, we predicted that miR-7a, a reported tumor suppressor miRNA which inhibits tumor migration and invasion, might be an indispensable player in the interplay between FADD and FAK (Table 1 and Figure 3A, 3B and 3C). [score:5]
Figure 5C shows the expression of miR-7a detected by qRT-PCR in B16F10 cells treated with miR-7a mimic and inhibitor. [score:5]
Cells overexpressed miR-7a migrated slower than its negative control while miR-7a knockdown provided a faster rate of migration compared to the miR-7a inhibitor negative control. [score:5]
Cells treated with siFADD scramble siRNA negative control (NC) and miR-7a mimic or inhibitor nc served as the sample control in the former experiment, while those treated with pRK5 empty vector plasmid and miR-7a mimic or inhibitor nc served as the sample control in the latter one. [score:5]
We increased miR-7a expression in murine B16F10 cells using miR-7a mimic and decreased it with miR-7a inhibitor. [score:5]
Our results revealed that the migration rate of B16F10 cells was significantly reduced when FADD expression was interfered by RNAi, but the migration rate was restored when cells were co -transfected with FADD siRNA and miR-7a inhibitor (Figure 7A). [score:5]
The results of miR-7a expression detected by qRT-PCR under these two conditions were shown in the Figure 3B and 3C, unraveling a negative relationship between miR-7a and FADD expression levels in MEF cells. [score:5]
H, I. and J. Representative western blot analysis (H) and quantification of expression of FADD (I) and FAK (J) in the miR-7a inhibitor or mimic co -transfected with pRK5-FADD experiment in B16F1s assessed by western blot. [score:5]
FADD deficiency inhibited FAK expression by promoting miR-7a in two murine melanoma cells with the same origin and genetic background but different metastatic potency, B16F10 and B16F1. [score:5]
Given that Keith M. Giles et al. [27] have identified miR-7 as a tumor suppressor miRNA which inhibits human melanoma cell migration and invasion, and considering the results mentioned above, we selected miR-7a for further analysis. [score:5]
As for the relationship between miR-7a and FAK, miR-7a could suppress FAK expression through binding to 3′UTR of FAK mRNA and lead to subsequent mRNA degradation. [score:5]
As previously mentioned, we demonstrated that expressions of FAK and FADD were positively correlated while the expression of miR-7a exhibited a negative correlation with these two proteins in MEFs. [score:5]
The postulated miR-7a target sequence of the murine FAK 3′ untranslated region (3′UTR) was 5′-GUCUUCC-3′, corresponding to nucleotides 749-755, as predicted by miRanda and PicTar. [score:5]
But when FADD expression was suppressed in these two cells, miR-7a's responses were sensitive in both B16F10 and B16F1 cells (shown in Figure 4C, 4D, 4G and 4H). [score:5]
A. and B. qRT-PCR and western blotting analyses of FAK expression in B16F10 cells treated with miR-7a mimic and inhibitor. [score:5]
Figure 3 A. Validation of expression of the only up-regulated miRNA, miR-7a, screened by microarray analysis in FADD [−/−] MEFs compared with FADD [+/+] MEFs (Table 1) detected by qRT-PCR. [score:5]
C. The expression level of miR-7a after treated by miR-7a mimic and inhibitor detected by qRT-PCR. [score:5]
A. Validation of expression of the only up-regulated miRNA, miR-7a, screened by microarray analysis in FADD [−/−] MEFs compared with FADD [+/+] MEFs (Table 1) detected by qRT-PCR. [score:5]
FADD overexpression increased the migratory ability of B16F1 cells which can be inhibited by miR-7a mimic (Figure 7B). [score:5]
Since B16F10 was highly metastatic with higher FAK expression than B16F1, we co -transfected FADD siRNA (siFADD) with either miR-7a mimic or inhibitor into B16F10 cells. [score:5]
Hsa-miR-7 (the same sequence as mmu-miR-7a) has been reported to inhibit EMT transition, metastasis and invasion in human breast, cervical cancer and glioblastoma cells via targeting focal adhesion kinase [28, 29, 36]. [score:5]
E, F. and G. Representative western blot analysis (E) and quantification of expression of FADD (F) and FAK (G) in the miR-7a inhibitor/mimic co -transfected with FADD siRNAs experiment in B16F10s assessed by western blot. [score:5]
Figure 5 A. and B. qRT-PCR and western blotting analyses of FAK expression in B16F10 cells treated with miR-7a mimic and inhibitor. [score:5]
FAK protein and mRNA levels were reduced in miR-7a mimic -transfected cells while upregulated in miR-7a inhibitor -transfected cells compared to each negative controls. [score:5]
We reasoned that miR-7a must bear a similar function by targeting FAK expression in melanoma as it does in other types of cancer. [score:5]
To sum up, these results suggest that miR-7a was an important mediator in FADD-regulated expression of FAK in both B16F10 and B16F1 cells. [score:4]
This study illustrates a potential role of FADD in murine melanoma cell migration by regulating FAK expression; a pathway that involves miR-7a as a crucial mediator. [score:4]
FADD regulated FAK and miR-7a expression in B16F10 and B16F1 melanoma cells. [score:4]
The mRNA expression of FAK decreased in cells with siFADD compared to NC group while the expression of miR-7a increased. [score:4]
After a 24 h period, we found that miR-7a overexpression substantially reduced the rate of cell migration, while miR-7a knockdown increased the rate of migration (Figure 6B). [score:4]
We suggest that FADD may play a novel role in cell migration by regulating FAK expression at which miR-7a acts as a mediator. [score:4]
E. FADD was overexpressed by transfected pRK5- FADD plasmid into B16F10 cells for 48 h. The mRNA expression of FAK increased in cells with pRK5-FADD plasmid compared to those with pRK5 empty vector plasmid while miR-7a decreased. [score:4]
To summarize, both in vitro and in vivo data suggest a crucial role of miR-7a in FADD-regulated expression of FAK and cell migration. [score:4]
Taken together, these data indicated that FAK is a miR-7a target in B16F10 murine melanoma cells. [score:3]
B. Firstly transfected pRK5-FADD plasmid alone or co -transfected pRK5-FADD plasmid together with miR-7a mimic or inhibitor into B16F1 cells for 48 hours. [score:3]
FAK was the target gene of miR-7a in murine melanoma B16F10 cells. [score:3]
A strong increase of miR-7a expression in FADD -deficient B16F1 and B16F10 cells was observed, as reported above. [score:3]
Therefore, it is reasonable to conclude that the miR-7a's response to FADD overexpression in B16F10 cells was not as sensitive as that in B16F1 cells. [score:3]
In contrast, cells co -transfected with FADD plasmid (pRK5-FADD) plus miR-7a inhibitor migrated remarkably faster than those transfected with FADD plasmids alone. [score:3]
A. Firstly, transfected FADD siRNA (siFADD) alone or co -transfected with FADD siRNA (siFADD) and miR-7a mimic or inhibitor into B16F10 cells for 48 hours. [score:3]
et al. [27] further announced miR-7 had a significant down-regulation in two human metastatic cell lines compared with the other primary melanoma-derived cell lines. [score:3]
Migration was retarded even more when miR-7a mimic were used instead of inhibitor co -transfected with FADD siRNAs. [score:3]
Mueller, D. W. et al. [2] demonstrated that miR-7 expression was decreased in human melanoma cells and associated with tumor metastasis. [score:3]
Figure 7 A. Firstly, transfected FADD siRNA (siFADD) alone or co -transfected with FADD siRNA (siFADD) and miR-7a mimic or inhibitor into B16F10 cells for 48 hours. [score:3]
The decrease of luciferase activities in cells co -transfected with functional FAK-3′ UTR plasmid and miR-7a mimic plasmid confirmed FAK is the target gene of miR-7a in melanoma as well. [score:3]
Mice injected with pRK5-FADD -transfected cells had more tumor nodules than those in the pRK5 group, whereas the number of tumor nodules seemed to be reduced by miR-7a mimic but increased by miR-7a inhibitor. [score:3]
The highly metastatic melanoma cell line B16F10 expresses high basic levels of FADD and FAK, low basic levels of miR-7a. [score:3]
B16F1 melanoma cells treated with pRK5 empty vector plasmids, pRK5-FADD plasmids, pRK5-FADD plasmids plus miR-7a mimic and pRK5-FADD plasmids plus miR-7a inhibitor, were collected and suspended in PBS. [score:3]
FAK was a target gene of miR-7a in B16F10 cells. [score:3]
We used wound healing assay to probe cell migratory ability under FADD and miR-7a knockdown or overexpression conditions. [score:3]
FADD overexpression promoted cell migration which is repressible by miR-7a mimic. [score:3]
It was not surprising that FAK expression was dramatically reduced compared to the cells treated with NC and miR-7a mimic, where the latter ones already exhibited decreased FAK expression compared to the two basic NC and miR-7a mimic nc. [score:3]
To verify our assumption, we transfected the mimic and inhibitor of miR-7a into B16F10 cells followed by analysis of mRNA and protein levels of FAK (Figure 5A and 5B). [score:3]
Cells co -transfected with pRK5-FADD and miR-7a inhibitor exhibited the fastest migration rate among the four different samples. [score:3]
FADD promoted cell migration in murine melanoma cells while miR-7a inhibited migration. [score:3]
MiR-7a was a mediator in FADD-regulated expression of FAK. [score:3]
Therefore it was reasonable to assume that FADD could have impact on cell migration via miR-7a targeting FAK signaling pathway in murine melanoma cells. [score:3]
As shown in Figure 7A, after a 24 h period, cells treated with siFADD migrated significantly slower than siRNA NC, and the migration of those treated with siFADD and miR-7a inhibitor was faster than siFADD. [score:3]
C57BL/6 mice were divided into 4 groups (5 animals/group): pRK5, pRK5-FADD, pRK5-FADD+miR-7a mimic and pRK5-FADD+miR-7a inhibitor. [score:3]
Conversely, the expression of miR-7a is lower in B16F10 cells than in B16F1. [score:3]
We employed programs miRanda and PicTar to predict target sites of miR-7a on FAK mRNA. [score:3]
On the other hand, pRK5-FADD plasmid was co -transfected with either miR-7a mimic or inhibitor into B16F1 cells. [score:3]
C. and D. B16F1 melanoma cells treated with pRK5 empty vector plasmids, pRK5-FADD plasmids, pRK5-FADD plasmids plus miR-7a mimic and pRK5-FADD plasmids plus miR-7a inhibitor, were collected and suspended in PBS. [score:3]
The decrease in miR-7a caused by artificial overexpression of FADD in B16F10 was not obvious compared to that in lowly metastatic cell line B16F1, leading to an inconspicuous change in FAK expression in B16F10 (Figure 4E and 4F), compared to a profound change in FAK in B16F1 (Figure 4I and 4J). [score:3]
siFADD plus miR-7a mimic conferred even more potent migratory inhibition than siFADD alone. [score:3]
We then monitored FAK expression of cells co -transfected with miR-7a mimic and FADD siRNA. [score:3]
A potential miR-7a target site was found at position 749-755 of the FAK 3′-UTR. [score:3]
FADD promoted cell migration in murine melanoma cells while miR-7a inhibited it. [score:3]
We injected the four groups of mice with B16F1 melanoma cells transfected with pRK5 empty vector plasmids, pRK5-FADD plasmids, pRK5-FADD plasmids plus miR-7a mimic, or pRK5-FADD plasmids plus miR-7a inhibitor through tail vein. [score:3]
Importantly, our work suggests that the FADD-miR-7a-FAK pathway can be a promising therapeutic target for melanoma cancer therapy. [score:3]
As shown from Figure 4C to Figure 4J, the qRT-PCR and western blotting assay indicated a positive correlation between FAK and FADD expression but a negative correlation between miR-7a and FADD in these two cells. [score:2]
The FADD/miR-7a/FAK pathway acted as a regulatory pathway of migration in murine melanoma cells, and this finding was consolidated by an in vivo mouse metastatic mo del. [score:2]
Both miR-7 and FAK have been reported to regulate melanoma migration and invasion, but the effects exerted by miR-7 and FAK in melanoma are opposite. [score:2]
To verify whether the predicted miR-7a binding sites in the 3′-UTR of FAK were responsible for the regulation, we cloned the original sequence of the predicted miR-7a binding site as well as a mutated version into a luciferase reporter vector (FAK-3′UTR and FAK-3′UTR-mutant) (Figure 5F). [score:2]
We have reasons to believe that miR-7a could be regulated by FADD at the transcription level and further efforts are required to unravel a more comprehensive mechanism. [score:2]
Expression of miR-7a, on the contrary, was reduced in B16F10 cells compared to B16F1 cells (Figure 4A). [score:2]
B. Migration analysis of B16F10 transfected with miR-7a mimic or inhibitor by wound healing assay at 24 hours. [score:2]
F. Alignments of nucleotides 749-755 in the FAK-3′UTR with mmu-miR-7a, and the mutation in the miR-7a -binding site of FAK-3′UTR-mut. [score:2]
In conclusion, we provide a new mo del of regulation of cell migration by the FADD-miR-7a-FAK pathway in murine melanoma cells. [score:2]
The relationship between FADD, FAK and miR-7a in murine melanoma cells. [score:1]
K. The systematic schema of the relations between FADD, FAK and miR-7a. [score:1]
Since both miR-7a and FAK were reported to be involved in cell migration in melanoma [27, 36, 37, 39], and our previous study demonstrated FADD's correlations with the two of them. [score:1]
As shown in Figure 5A, miR-7a mimic significantly reduced the mRNA level of FAK. [score:1]
And another vector named pGL-FAK-3′UTR-mutant was generated from pGL-FAK-3′UTR by deleting the putative binding site for miR-7a “GUCUUCC”. [score:1]
Here we also confirmed the anti-migratory function of miR-7a in murine melanoma cells. [score:1]
Firstly, we measured the basic expression of FADD, FAK and miR-7a in these two murine melanoma cells. [score:1]
miR-7 prohibits migration while FAK promotes it [27, 36– 38]. [score:1]
Therefore, we assumed a similar role of miR-7a in murine melanoma cells. [score:1]
The sequences of siRNA and miRNAs are as follows: FADD: 5′-ACGAUCUGAUGGAGCUCAA-3′; miR-7a (MIMAT0000677): 5′-UGGAAGACUAGUGAU UUUGUUGU-3′. [score:1]
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[+] score: 401
Thus, it could be speculated that although miR-7a expression was induced after MI injury, the synergistical increase of Cdr1as level could reduce miR-7a activity by sponging miR-7a, leading to the upregulated expression of miR-7a targets, including PARP and SP1, which play pro-apoptotic roles during MI development. [score:11]
Moreover, Cdr1as overexpression in vivo increased cardiac infarct size with upregulated expression of PARP and SP1, while miR-7a overexpression reversed these changes. [score:10]
Previous study also showed that miR-7a expression is upregulated by unfolded protein response (UPR) associated with MI development, and simulated in vitro ischemia in cardiomyoblasts [23], while ectopic expressed miR-7a provides resistance against UPR -mediated apoptosis. [score:9]
Indeed, the upregulation of SP1 and PARP was also observed in Cdr1as overexpressing cardiomyocytes, indicating the role of Cdr1as, as one important regulator to prevent miR-7a from interacting with its target transcripts. [score:9]
Since Cdr1as acted as an apoptosis inducer in cardiomyocytes, and was also proven to induce the upregulation of PARP and SP1 probably by sponging miR-7a in vitro, the overexpression of Cdr1as was performed in vivo through the transfection of pcDNA-Cdr1as via intracardial injection to verify whether Cdr1as functioned through upregulating PARP and SP1 in MI mice. [score:9]
In addition, Cdr1as overexpression in vivo could aggravate MI development with increased cardiac infract size, as well as strongly upregulated PARP and SP1, while miR-7a overexpression significantly attenuated these Cdr1as -induced changes. [score:9]
Nevertheless, Cdr1as -induced effects could all be reversed by the overexpression of miR-7a through the co-transfection of lentivirus with miR-7a mimic (Fig 6), supporting that miR-7a could protect cardiomyocytes from Cdr1as -induced aggravation of MI injuries, involving the re -downregulated expression of PARP and SP1. [score:8]
Overexpression of Cdr1as Aggravated MI Injuries with Upregulated PARP and SP1, which Could Be Rescued by the Co-Overexpression of miR-7a. [score:8]
Moreover, the overexpression of Cdr1as was proven to induce developmental defects in the midbrain of embryonic zebrafish, similar to that induced by miR-7 inhibition, while miR-7 overexpression could partially rescue the Cdr1as -induced phenotype [19]. [score:8]
Given the fact that miR-7 overexpression shows regulation on stress -induced apoptosis of cardiomyocytes [21, 23], it is possible that ectopic Cdr1as expression may also influence cardiomyocyte apoptosis due to their co -expression. [score:8]
The SP1 was identified as a new miR-7a target, in line with previously identified PARP, while miR-7a -induced decrease of cell apoptosis under hypoxia treatment was proven to be inhibited by PARP-SP1 overexpression. [score:7]
MiR-7a Targeted the Expression of PARP and SP1, which Were Upregulated by Cdr1as. [score:7]
Our previous study reported the upregulated expression of miR-7a/b in murine hearts with myocardial ischemia-reperfusion (I/R) injury [21], while miR-7a/b functioned in protecting myocardial cells against I/R -induced apoptosis by negative regulation on poly(ADP-ribose) polymerase (PARP), an executioner of apoptosis [22]. [score:7]
Effect of Cdr1as overexpression on MI -induced infarct size (A), area at risk (B), lactate dehydrogenase (LDH) release (C), and the expression of PARP and SP1 (D; bar = 100μm) was analyzed, as well as miR-7a co-overexpression with Cdr1as through the delivery of miR-7a mimic three days before MI introduction. [score:7]
In order to verify this speculation, the effects of miR-7a and its targets, PARP and SP1, on hypoxia -induced apoptosis were analyzed in cardiomyocytes under the overexpression of miR-7a and the subsequent co-overexpression of PARP and SP1. [score:7]
These results implied that the Cdr1as overexpression might regulate cardiomyocyte apoptosis by reducing miR-7a activity, due to its regulation on apoptosis-related targets. [score:7]
Therefore, the circular RNA Cdr1as also functioned as a miR-7a sponge in promoting MI injuries by reduced the activity of miR-7a in inhibiting the expression of its target genes, PARP and SP1. [score:7]
To elucidate the molecular mechanisms of miR-7a in cardiomyocyte apoptosis after MI injury, putative targets involving the apoptotic program were screened by using TargetScan, one microRNA target prediction program (http://www. [score:7]
Additionally, the expression levels of endogenous miR-7a were detected to be not induced by Cdr1as overexpression, while miR-7a overexpression alone also showed little effect on the apoptosis of normal cultured cardiomyocytes (data not shown), in line with our previous studies [21]. [score:7]
In addition, the expression of PARP and SP1 at both mRNA and protein level were detected to be inhibited significantly in miR-7a overexpressing cells (Fig 4C and 4D). [score:7]
Firstly, Cdr1as overexpression plasmid (pcDNA-Cdr1as) or miR-7a mimic was transfected into MCM cells for 48h, and Cdr1as or miR-7a expression was confirmed to be increased by around 3.3-fold or ~22-fold respectively, as compared to the control (Fig 3A and 3B), while the expression levels of endogenous miR-7a showed no obvious changes under pcDNA-Cdr1as transfection (Fig 3B). [score:6]
Upregulated expression of Cdr1as and miR-7a was detected in myocardial tissues from MI mice. [score:6]
According to previous studies, the co -expression of Cdr1as and miR-7a indicate potential interaction between them, wherein Cdr1as can function as a miR-7 inhibitor, buffer or reservoir under different conditions by sponging miR-7, showing regulation on miR-7 function [20]. [score:6]
In the current study, the predicted mo-miR-7a binding sites at the 3’ UTR of PARP were also observed through bioinformatics analysis, while further luciferase assays and expression detection under miR-7a overexpression demonstrated it to be one conserved miR-7a target. [score:6]
After the transfection of miR-7a mimic or negative control microRNA (Pre-NC) into MCM cells, luciferase reporters were co -transfected for 48 h. showed that miR-7a strongly inhibited luciferase activity of the GV126-SP1 3’UTR -WT, but not the GV126-SP1 3’UTR -MU (P < 0.01, Fig 4B), indicating its direct target on SP1. [score:6]
Regulation of miR-7a on the expression of PARP and SP1 was inhibited by Cdr1as. [score:6]
Further results revealed the mechanism of the Cdr1as/miR-7a pathway in MI injury, wherein Cdr1as acted as a miR-7a sponge by inhibiting the regulation of miR-7a on its targets. [score:6]
However, miR-7a upregulation by simulated I/R or MI showed a conflict with its protective role against stress -induced apoptosis in cardiomyocytes, indicating an unrevealed regulatory mechanism needed to be clarified. [score:5]
These results also implied that miR-7a activity, in protecting against cardiomyocytes apoptosis as indicated previously [21, 23], was inhibited by Cdr1as overexpression. [score:5]
To verify whether miR-7a and its targets, PARP and SP1, potentially functioned in the apoptotic process during MI injury, miR-7a overexpressing MCM cells were subjected to hypoxia treatment, and cell apoptosis with activated caspase-3 was analyzed accordingly (Fig 5C), as well as the percentage of apoptotic cells (Fig 5D). [score:5]
Overexpression of miR-7a Protected MCM Cells from Hypoxia-Induced Cell Apoptosis by Targeting PARP and SP1. [score:5]
The mouse myocardial infraction mo del was successfully established with observed enlargement of infarct size, increase of AAR and LDH release, and the expression of Cdr1as and miR-7a were both detected to be significantly upregulated in myocardial tissues of MI mice as compared to the control. [score:5]
These results illustrated that miR-7a protected cardiomyocytes from hypoxia -induced apoptosis by inhibiting the function of its targets, PARP and SP1. [score:5]
showed that hypoxia -induced apoptosis was inhibited by miR-7a, but was then reversed by the co-overexpression of PARP and SP1. [score:5]
Cdr1as expression was elevated in hypoxia -treated cardiomyocytes with overexpressed miR-7a. [score:5]
Cdr1as overexpression in MCM cells promoted cell apoptosis, but was then reversed by miR-7a overexpression. [score:5]
Further results showed that hypoxia -induced apoptosis could be recovered by the co-overexpression of PARP and SP1, through the co-transfection of pcDNA-PARP-SP1 into miR-7a overexpressing cells (Fig 5C and 5D). [score:5]
Then, the caspase-3 activity, one common used detector for apoptosis, and the percentage of apoptotic cells were analyzed in cells with Cdr1as overexpression or co-overexpression of Cdr1as and miR-7a. [score:5]
These observations explain why upregulated miR-7a showed cardiomyocyte protection during MI injury. [score:4]
Similarly, co -upregulated Cdr1as and miR-7a were confirmed in hypoxia -treated cardiomyocytes, as simulation of MI in vitro. [score:4]
Cdr1as also functioned as a powerful miR-7a sponge in myocardial cells, and showed regulation on the protective role of miR-7a in MI injury, involving the function of miR-7a targets, PARP and SP1. [score:4]
Cdr1as and miR-7a were both upregulated in MI mice with increased cardiac infarct size, or cardiomyocytes under hypoxia treatment. [score:4]
Since the potential role of Cdr1as as a miR-7 sponge has been revealed in the mouse brain [18], it was hypothesized that Cdr1as may also be co -overexpressed with miR-7a in cardiomyocytes during MI -induced injury. [score:3]
Overexpression of miR-7a Protected Cardiomyocyte from Cdr1as-Induced Apoptosis. [score:3]
It is important to note that SP1 was proven to be a new functional target of miR-7a involved in miR-7a mediated cardiomyocytes protection against MI injury. [score:3]
0151753.g003 Fig 3MCM cells were transfected with pcDNA-Cdr1as or miR-7a mimic for the overexpression of Cdr1as and miR-7a respectively, with pcDNA or Pre-NC as the respective negative control. [score:3]
These studies supported the key role of Cdr1as as a miR-7 sponge or inhibitor by binding miR-7 seed sites. [score:3]
showed that Cdr1as -induced apoptosis, with increased caspase-3 activity, could be reversed by miR-7a co-overexpression. [score:3]
Therefore, in this study, Cdr1as was speculated to be co-expressed with miR-7a in cardiomyocytes after MI injury. [score:3]
Hypoxia treatment induced significant activation of caspase-3 with increased apoptotic cells, while the overexpression of miR-7a could rescue these apoptosis phenotypes induced by hypoxia. [score:3]
Recent studies have demonstrated the role of Cdr1as (or CiRS-7), one of the well-identified circular RNAs (circRNAs), as a miR-7a/b sponge or inhibitor in brain tissues or islet cells. [score:3]
Notably, SP1, a member of the SP/KLF family of transcription factors [34], was also identified as one target gene of miR-7a. [score:3]
Additionally, the expression of Cdr1as and miR-7a showed a respective 2.4-fold and 1.9-fold increase in cardiomyocytes at 24 h after MI surgery (P < 0.01; Fig 1D and 1E). [score:3]
These results illustrated that Cdr1as indeed co-expressed with miR-7a in myocardial cells. [score:3]
In the mouse brain, particularly in the neocortical and hippocampal neurons, Cdr1as and miR-7 were observed to be co-expressed [18]. [score:3]
Moreover, the co -expression of Cdr1as and miR-7a was also confirmed in both primary cardiomyocytes from normal CL57B6 mice and MCM cells with a time dependent manner after hypoxia treatment for simulating MI injury in vitro (Fig 2). [score:3]
Further functional studies were performed to illustrate the potential mechanism of the Cdr1as/miR-7a pathway during MI -induced apoptosis, including the exploration of new miR-7a target genes related to the apoptotic process. [score:3]
C. RT-PCR analysis of mRNA level of PARP and SP1 under miR-7a or Cdr1as overexpression. [score:3]
To confirm whether miR-7a targeted SP1 through its 3’UTR, a firefly luciferase reporter plasmid containing the 3’UTR segment of SP1 mRNA with the putative miR-7a binding sites was constructed (GV126-SP1 3’UTR-WT), while another mutant luciferase reporter vector containing the mutated binding sites (GV126-SP1 3’UTR-MU) was used as negative control. [score:3]
Real-time PCR was performed to quantify the expression of Cdr1as and miR-7a in cardiomyocytes. [score:3]
B. Luciferase activity of the 3’-UTR of PARP and SP1 in miR-7a overexpressing cells, after normalized to that of Renilla. [score:3]
The 3’ untranslated region (UTR) sequence of SP1 or PARP containing the predicted mo-miR-7a binding sites was amplified from MCM genome and sub cloned into the multiple cloning sites in the downstream of the luciferase gene in the GV126 vector (Promega Corporation, Durham, NC, USA), resulting in the wild-type luciferase reporter construct, GV126-SP1-3’UTR-WT or GV126-PARP-3’UTR-WT. [score:3]
of Cdr1as overexpressing plasmid and miR-7a mimic were conducted for gain-of-function studies. [score:3]
Besides, hypoxia -induced apoptosis could also be reversed by the transfection of PARP or SP1 siRNA (data not shown), similar to the transfection of miR-7a mimic, indicating that miR-7a protected cardiomyocytes from hypoxia -induced cell apoptosis by targeting PARP and SP1. [score:3]
B. Expression of miR-7a. [score:3]
As shown in Fig 3C and 3D, Cdr1as significantly increased the caspase-3 activity and apoptotic cells, while co-overexpression of miR-7a reversed Cdr1as -induced phenotypes, resulting in reduced caspase-3 activity and apoptotic cells. [score:3]
Hypoxia -induced apoptosis was mediated by the targeting of miR-7a on PARP and SP1. [score:3]
MCM cells were transfected with pcDNA-Cdr1as or miR-7a mimic for the overexpression of Cdr1as and miR-7a respectively, with pcDNA or Pre-NC as the respective negative control. [score:3]
Our previous study also demonstrated that PARP is a functional target gene of miR-7a/b involved in miR-7a/b—mediated cardiomyocyte protection against I/R in a rat mo del [21]. [score:3]
And, myocardial tissues were disassociated at 6, 12, or 24 h after MI introduction, to detect the expression of Cdr1as (D) and miR-7a (E) by qRT-PCR analysis. [score:3]
After the transfection, Cdr1as or miR-7a expression level was confirmed by qRT-PCR. [score:3]
Co-Expression of Cdr1as and miR-7a Was Detected during MI-Induced Injury or in Hypoxia-Treated Cardiomyocytes. [score:3]
For miR-7a expression, microRNA was analyzed by using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) with provided RT-U6 and microRNA-specific stem-loop primers, and the expression levels were determined through TaqMan MicroRNA assays with the TaqMan Universal PCR Master Mix (Applied Biosystems). [score:3]
Thus, in order to analyze the potential effects of Cdr1as on miR-7a function, the overexpression of Cdr1as and miR-7a was performed successively in a murine myocardial cell line (MCM). [score:3]
Recently, one human circRNA, antisense to the cerebellar degeneration-related protein 1 transcript (Cdr1as) [17], also termed as ciRS-7 (circular RNA sponge for miR-7) [18], has been identified to share expression domains with miR-7 [19]. [score:3]
However, the potential role of Cdr1as in myocardial cells has not yet been explored although miR-7a has been reported to be overexpressed in stress-stimulated cardiomyocytes [21, 23]. [score:3]
Thus, it was suggested that miR-7a might only function in regulating stress -induced apoptosis of myocardial cells. [score:2]
MiR-7a overexpression reversed Cdr1as -induced bad effects in mouse MI mo del. [score:2]
In this study, SP1 was also validated as a new miR-7a target in cardiomyocytes through luciferase assays and gain-of-function studies. [score:2]
MiR-7a overexpression reversed Cdr1as -induced apoptosis of MCM cells. [score:2]
Luciferase reporter assay and western blot analysis were performed to verity potential miR-7a targets. [score:2]
These results indicated the presence of a Cdr1as/miR-7a pathway in murine cardiomyocytes wherein the potential function of miR-7a in regulating cardiomyocyte apoptosis might also be mediated by Cdr1as. [score:2]
And, under different mechanisms, Cdr1as might also act as a miR-7 buffer or reservoir in modulating miR-7 function [20]. [score:1]
Therefore, it can be speculated that SP1 also functions in miR-7a mediated cardiomyocyte apoptosis during MI, in line with PARP. [score:1]
Thus, in the present study, the presence of the Cdr1as/miR-7a pathway was characterized by detecting their co -expression in cardiomyocytes with MI induction, to explore whether Cdr1as showed similar negative effects on miR-7a function in cardiomyocytes. [score:1]
Circular RNA and miR-7 in cancer. [score:1]
Cells transfected with Pre-NC (for miR-7a mimic) or pcDNA (for pcDNA-Cdr1as) were used as control D. Western blot analysis of protein level of PARP and SP1. [score:1]
A. Conserved miR-7a binding sites in 3’-UTR of PARP and SP1. [score:1]
Similar analysis for PARP, another miR-7a target that has been demonstrated to function in I/R -induced apoptosis in our previous study [21], was also performed here for further investigation (Fig 4A and 4B). [score:1]
The circular RNA Cdr1as is derived from an antisense transcript of the CDR1 protein-coding gene at chromosome X (NC_000086.7) in mice [17], containing binding sites or clusters for binding miR-7, and function as a miR-7 sponge in neuronal cells [18], or in newly reported islet cells [26]. [score:1]
In conclusion, this study illustrated the presence of the Cdr1as/miR-7a axis in cardiomyocytes for the first time. [score:1]
Lentiviral vectors carried miR-7a mimic (miR-7a mimic) and its scrambled control microRNA (Pre-NC) were obtained from Genepharma (Shanghai, China), as well as corresponding lentivirus (1×10 [9] TU/ml) for in vivo studies. [score:1]
SP1, one transcription factor implicated in hypoxic gene transcription [30], was found to behave conserved binding sites for miR-7a (Fig 4A). [score:1]
Conserved miR-7a binding sites in 3’-UTR of PARP and SP1. [score:1]
MCM cells were transfected miR-7a mimic or co -transfected with miR-7a mimic and pcDNA-PARP-SP1, and then subjected to hypoxia treatment. [score:1]
0151753.g005 Fig 5MCM cells were transfected miR-7a mimic or co -transfected with miR-7a mimic and pcDNA-PARP-SP1, and then subjected to hypoxia treatment. [score:1]
Moreover, miR-7a showed potential therapeutic value for improving MI-related injuries. [score:1]
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[+] score: 368
Other miRNAs from this paper: hsa-mir-7-1, hsa-mir-7-2, hsa-mir-7-3, mmu-mir-7a-1, mmu-mir-7b
Thus, our work showed for the first time that TTF-1-promoter-operating miR-7 expression might be an ideal strategy for targeted expression of miR-7 in lung cancer, which provides preliminary experimental basis for targeted expression of distinct miRNAs in lung cancer and was helpful for the development of gene therapy against clinical lung cancer. [score:12]
15, 49, 50 To reach a comprehensive knowledge on the molecular mechanism of the effect of TTF-1-operating miR-7 expression on the growth and metastasis of lung cancer cells, we used a global gene expression chip technique combined with biological information analysis to screen the potential target of miR-7. Unexpectedly, we found that TTF-1-operating miR-7 expression could significantly reduce the expression level of NDUFA4 in lung cancer cells in vitro and in vivo. [score:11]
Luckily, we found that this plasmid could effectively express miR-7 in human lung cancer cells with a higher expression level of TTF-1 in vitro, but not other human cancer cells with a lower expression level of TTF-1, including colon cancer, hepatocellular carcinoma, and gastric cancer, indicating that the TTF-1 promoter could effectively orchestrate targeted expression of miR-7 in lung cancer cells. [score:11]
To further elucidate the potential molecular mechanism through which TTF-1-promoter-operating miR-7 expression affected the growth of lung cancer cells, we used miRBase and TargetScan software to compare the downregulated genes in the p-T-miR-7 injection group and found 11 putative miR-7 target genes, including NXT2, C5orf22, PIGH, NDUFA4, TMEM97, CHAMP1, CNN3, LRRC8B, SAYSD1, TRMT13, and TPGS2 (Figure 4D), which also were closely related to tumor cell growth according to previous literatures. [score:10]
Of note, we found that overexpression of NDUFA4, a novel target molecule of miR-7, could abrogate the effect of TTF-1-promoter-operating miR-7 expression on the growth of lung cancer cells, accompanied with altered expression of phosphorylation of Akt and Erk. [score:9]
However, when we further analyzed the expression level of miR-7, it was found that, compared with their corresponding control, miR-7 level increased significantly in heart, spleen, lung, intestine, and lymph nodes, respectively (Figure S3), indicating that intrinsic transcriptional factors in these organs and tissues might bind to TTF-1 promoter and manipulate the expression of miR-7. It is the first time the potential value of the TTF-1 promoter operating distinct miRNAs expression in targeted gene therapy against lung cancer has been explored. [score:8]
In conclusion, for the first time, our study demonstrated that TTF-1-promoter-operating miR-7 expression could significantly inhibit the growth of human lung cancer in vitro and in vivo, closely related to downregulation of NDUFA4 and altered transduction of related signaling pathways including Akt and Erk. [score:8]
However, when we further analyzed the expression level of miR-7, it was found that, compared with their corresponding control, miR-7 level increased significantly in heart, spleen, lung, intestine, and lymph nodes, respectively (Figure S3), indicating that intrinsic transcriptional factors in these organs and tissues might bind to TTF-1 promoter and manipulate the expression of miR-7. To investigate whether the TTF-1 promoter might be an ideal candidate for operating miR-7 expression in lung cancer, as shown in Figure 1A, we first amplified and inserted the sequence of both TTF-1 promoter and miR-7 into pGL3.0 basic vector and successfully constructed an eukaryotic expression vector, which could express miR-7 operated by TTF-1 promoter (termed as p-T-miR-7) (Figure S1). [score:8]
Then, to further explore whether downregulation of NDUFA4 contributed to the suppressive effect of TTF-1-promoter-operating miR-7 expression on human lung cancer cells, we transiently co -transfected p-T-miR-7 and p-NDUFA4 into lung cancer cell line 95D cells and observed the possible change on cell growth and metastasis. [score:8]
Notably, we further revealed that the downregulation of NDUFA4, a novel target of miR-7, contributed to the effects of miR-7 expression operated by TTF-1 promoter on the growth and metastasis of human lung cancer cells, accompanied by altered transduction of related signaling pathway including the Akt and Erk pathway. [score:8]
Collectively, our data indicated that TTF-1-promoter-operating miR-7 expression could affect the growth of tumor cells, which might be closely due to the downregulation expression of NDUFA4. [score:8]
[17] Moreover, the reduced expression of miR-7 was associated with the sites mutation of its promoter region in lung cancer tissues, indicating that miR-7, an important tumor suppressor, could be used as candidate for targeted gene therapy against lung cancer. [score:8]
These data indicated that TTF-1-promoter-operating miR-7 expression might be an ideal strategy in lung cancer, which provided preliminary experimental basis for targeted expression of distinct miRNA in lung cancer and was helpful for the development of gene therapy against clinical lung cancer. [score:8]
Combining these data demonstrated that miR-7 could be effectively targeted expression by the TTF-1 promoter and suppressed the growth of human lung cells in vitro. [score:7]
Therefore, successive research work on both the potential effect of TTF-1-promoter-operating miR-7 expression on other types of cancers and regulatory factors including transcript factors in the activation of TTF-1 promoter, which did not been investigated in present study, is valuable for the verification of usage of the TTF-1 promoter in targeted gene expression in lung cancer and can ultimately benefit the development of a therapeutic strategy in clinical lung cancer. [score:7]
Unexpectedly, real-time PCR assay showed that only NDUFA4, one target among all predicted target genes of miR-7, was significantly downregulated more than five times both in tumor tissue in the p-T-miR-7 injection group (Figure 4E; p < 0.05) and in p-T-miR-7 -transfected tumor cells, respectively (Figure 4F; p < 0.05). [score:7]
Overexpression of NDUFA4 Abrogated the Suppressive Effect of TTF-1-Promoter-Operating miR-7 Expression. [score:7]
17, 30 In order to explore the underlying mechanism of TTF-1-promoter-operating miR-7 expression on the growth of lung cancer cells, we analyzed the global gene expression profile in tumor tissue between the p-T-miR-7 and the p-Cont injection group using gene expression microarray assay. [score:6]
Moreover, the metastatic index of lung also decreased significantly (Figure 2J; p < 0.05), indicating TTF-1-promoter-operating miR-7 expression also could significantly inhibit the metastasis of lung cancer cells in vivo, which was consistent with our previous report. [score:5]
Importantly, we further found that TTF-1-promoter-operating miR-7 expression could significantly not only inhibit the growth and metastasis of human lung cancer cells in vivo, but also induce the apoptosis of cancer cells in vivo. [score:5]
Importantly, overexpression of NDUFA4 could abrogate the effect of TTF-1-operating miR-7 expression on the growth and metastasis of lung cancer cells, accompanied by altered transduction of the Akt and Erk pathway, which was critical for the growth and metastatic potential of lung cancer cells. [score:5]
TTF-1-Promoter-Operating miR-7 Expression Suppressed the Growth of Human Lung Cells In Vitro. [score:5]
Importantly, we found that TTF-1-promoter-operating miR-7 expression could effectively inhibit the growth of lung cancer cells in vitro and in vivo. [score:5]
It is well known that miR-7, as an intrinsic tumor suppressor, has been found to be an important regulator in the development of various cancers including lung cancer. [score:5]
All of the above data demonstrated that TTF-1 promoter could effectively operate miR-7 expression in tumor mass, which subsequently inhibited tumorigenicity of lung cancer in vivo. [score:5]
Importantly, we analyzed the expression level of miR-7 in tumor tissue and found that the expression level of miR-7 in the p-T-miR-7 injection group was significantly higher than that in the p-Cont injection group (Figure 2G; p < 0.05). [score:5]
TTF-1-Promoter-Operating miR-7 Expression Reduced the Expression of NDUFA4. [score:5]
These data demonstrated that NUDFA4, a novel target of miR-7, contributed to the effects of TTF-1-operating miR-7 expression in lung cancer cells. [score:5]
For example, Xiong et al. [15] found that overexpression of miR-7 suppressed NSCLC cells proliferation and migration in vitro, and reduced tumorigenicity in vivo. [score:5]
[17] Thus, to further verify the effect of TTF-1-promoter-operating miR-7 expression on the growth of lung cancer cells, we analyzed the expression of phosphorylation of Akt and Erk in tumor tissue derived from the p-T-miR-7 injection group or the p-Cont injection group, respectively. [score:5]
Importantly, remote hypodermic injection, but not local injection, of plasmid p-T-miR-7 could significantly inhibit the growth and metastasis of human lung cancer cells in vivo, accompanied by altered expression of growth- and metastasis -associated molecules such as CDK and MMP family members. [score:5]
12, 13, 14 For lung cancer, accumulating evidence suggested that miR-7 was an important regulator in the development of lung cancer through controlling the growth and invasion, as well as apoptosis, of lung cancer cells and emerged as a novel potential therapeutic target. [score:5]
At the same time, Li et al. [16] showed that restoration of miR-7 expression suppressed the tumorigenicity of lung cancer cells in vivo. [score:5]
Finally, to confirm the effect of TTF-1-promoter-operating miR-7 expression on the growth and metastasis of tumor cells in vivo, we further detected the expression of cell-growth-related molecules including CDK2, CDK3, CDK4, and CDK6, as well as metastasis-related molecules including CXCR4, E-Cadherin, MMP2, MMP3, and MMP9, in tumor mass, respectively. [score:5]
Next, to observe the target efficiency, we further transfected the plasmid p-T-miR-7 into six different human cancer cell lines, including lung cancer cell line 95D cells, A549 cells, NCI-H292 cells, gastric cancer cell line SGC901 cells, hepatitic cancer cell line HepG2 cells, and colon cancer cell line SW620 cells, and then detected the expression level of miR-7 operated by TTF-1 promoter. [score:5]
1, 12, 13 Moreover, our previous research works also showed that overexpression of miR-7 could inhibit the growth of human lung cancer cells. [score:5]
To investigate whether the TTF-1 promoter might be an ideal candidate for operating miR-7 expression in lung cancer, as shown in Figure 1A, we first amplified and inserted the sequence of both TTF-1 promoter and miR-7 into pGL3.0 basic vector and successfully constructed an eukaryotic expression vector, which could express miR-7 operated by TTF-1 promoter (termed as p-T-miR-7) (Figure S1). [score:5]
TTF-1-Promoter-Operating miR-7 Expression Inhibited Tumorigenicity In Vivo. [score:5]
24, 25, 26 Accumulating literatures have documented that miR-7, as a promising tumor suppressor, could regulate the biological behavior of human lung cancer cells. [score:4]
Real-time PCR assay showed that the expression level of the miR-7 in p-T-miR-7 transfection group was unmistakably higher than that in the control group (Figure 1B; p < 0.05), indicating TTF-1 promoter could effectively operate the expression of miR-7 in lung cancer cells. [score:4]
[18] Interestingly, some research works showed that in situ local injection of miR-7 overexpression plasmids could regulate the growth and metastatic potential of human lung cancer cells in vivo via the Akt pathway. [score:4]
And our recent work reported that the site mutation of miR-7 promoter region contributed to its altered expression in clinical lung cancer tissue. [score:4]
24, 25, 26Accumulating literatures have documented that miR-7, as a promising tumor suppressor, could regulate the biological behavior of human lung cancer cells. [score:4]
Therefore, combining these research works may highlight the fact that miR-7 is a critical regulator and might be an ideal target for gene therapy against clinical lung cancer, which would be helpful for the outcome of clinical treatment. [score:4]
Data showed that there were not any changes on the expression level of both Akt and Erk in between the p-T-miR-7 injection group and the p-Cont injection group. [score:3]
In order to test the efficiency of TTF-1-promoter-operating miR-7 expression, we then transiently transfected plasmid p-T-miR-7 into human lung cancer cell line 95D cells. [score:3]
Finally, we also analyzed the expression of phosphor-Akt and phosphor-Erk in the p-T-miR-7 and p-NDUFA4 co-transfection groups. [score:3]
43, 44 In the present study, we further extended previous findings to report that expression of miR-7, operated by TTF-1 promoter, could reduce the growth and metastasis of human lung cancer cells in vitro. [score:3]
Thereby, further studies on the dynamic distribution of the plasmid and the possible change on other organs are important for the evaluation of safety of the plasmid in vivo, which is critical for the potential application of gene therapy based on targeted miR-7 expression in lung cancer. [score:3]
However, the expression level of phosphor-Akt and phosphor-Erk were decreased significantly in the p-T-miR-7 injection group (Figures 3A and 3B; p < 0.05). [score:3]
We first observed that TTF-1 promoter could effectively operate miR-7 expression in lung cancer cells. [score:3]
27, 28, 29 Our previous work also showed that miR-7 could inhibit the proliferation and metastasis of lung cancer cells though the Akt pathway. [score:3]
13, 39, 40, 41 For example, Xiong et al. [42] documented that miR-7 could inhibit the growth of human lung cancer cells in vivo. [score:3]
To confirm these data, we further detected the expression of miR-7 in tumor mass by in situ hybridization and obtained a similar result (Figure 2H). [score:3]
TTF-1-Promoter-Operating miR-7 Expression Altered the Transduction of the Akt/Erk Pathway. [score:3]
Therefore, in this present study, we first constructed an eukaryotic vector of promoter of TTF-1-gene-operating expression of miR-7 (termed as p-T-miR-7) and observed its effects on the growth and migration of human lung cancer cells in vitro. [score:3]
Next, we further monitored the impact of TTF-1-promoter-operating miR-7 expression on lung cancer cells in vivo. [score:3]
Data showed that the expression of all of these molecules in the p-T-miR-7 injection group decreased unmistakably (Figure 2l; p < 0.05). [score:3]
The altered gene expression profiles in p-T-miR-7 were shown in a heatmap (Figures 4A and 4B). [score:3]
Interestingly, we found that the expression level of miR-7 was higher in lung cancer cells, including 95D cells, A549 cells, and NCI-H292 cells, than in other types of tumor cells (Figure 1C; p < 0.05), indicating higher intrinsic activity of TTF-1 promoter in lung cancer cells. [score:3]
To further explore the potential therapeutic effect of TTF-1-promoter-operating miR-7 expression on tumorigenicity in vivo, a xenograft mo del of human lung cancer in nude mice was adopted. [score:3]
Combining these results demonstrated that TTF-1-promoter-operating miR-7 expression affected the growth and metastasis of human lung cancer cells through NDUFA4. [score:3]
Finally, we observed that there were not any significantly change in various important organs or tissues, indicating the safety value of strategy of TTF-1-promoter-operating expression of miR-7 in vivo. [score:3]
Consistently, our previous work also showed that overexpression of miR-7 could reduce the growth and metastasis of human lung cancer cells in vivo and in vitro. [score:3]
These results suggested that TTF-1-promoter-operating miR-7 expression attenuated the growth of human lung cells in vitro and in vivo by altering the transduction of the Akt/Erk pathway, which was consistent with our previous findings. [score:3]
The plasmid p-T-miR-7 or p-Cont (100 mg) was given locally by direct injection into the left flank of nude mice five times every 3 days. [score:2]
As shown in Figure 1F, the relative expression of these CDK family members also decreased significantly in the p-T-miR-7 -transfected group compared with those in the control group (p < 0.05). [score:2]
Accumulating evidence showed that the molecular mechanism through which miR-7 regulated the growth and metastasis of lung cancer cells was complex, and various genes including PA28 gamma, KLF4, BCL-2, and so on also were reportedly involved in the biological function of miR-7 in lung cancer. [score:2]
To confirm these findings, we also transiently transfected p-T-miR-7 or p-Cont into lung cancer cell line 95D cells in vitro, respectively, and found the expression level of both phosphor-Akt and phosphor-Erk were decreased significantly in the p-T-miR-7 -transfected group compared with those in the p-Cont -transfected group (Figures 3C and 3D; p < 0.05). [score:2]
An increasing body of literatures documented that miR-7 could regulate the growth of lung cancer cells though various signal pathways such as the Akt and Erk pathway. [score:2]
Moreover, we further evaluated the potential effect of the TTF-1-promoter-operating miR-7 expression on the growth and metastasis of human lung cancer cells in vivo. [score:1]
[54] Different from these research works, we analyzed the distribution of plasmid p-T-miR-7 after remote hypodermic injection and found that the plasmid was dominantly enriched in lung tissue and tumor mass in vivo. [score:1]
As shown in Figures 6A and 6B, the proliferation of 95D cells decreased significantly in the p-T-miR-7 transfection group, which was consistent with our above data. [score:1]
20, 21, 22 Therefore, in the present study, we attempted to design and construct an eukaryotic vector encoding miR-7, which was manipulated by the TTF-1 promoter. [score:1]
miR-7 TTF-1 95D cells lung cancer growth NDUFA4 Lung cancer is one of the most common causes of cancer death. [score:1]
After blocking with normal goat serum (1:100), sections were next incubated or microwave heating and then incubated with hybridization cocktail containing miR-7 probe (1:1,000 dilution; EXIQON; no. [score:1]
Notably, we found that the proliferation of cells in the p-T-miR-7 and p-NDUFA4 co-transfection group elevated unmistakably (p < 0.05). [score:1]
Finally, we preliminarily estimated the distribution of plasmid p-T-miR-7 in vivo. [score:1]
7 days later, the plasmid of p-T-miR-7 or p-Cont was remotely given by subcutaneous injection into the left flank of nude mice five times every 3 days (Figures 2A and 2B). [score:1]
As shown in Figure 1D, the proliferation of human lung cancer cell line 95D cells decreased significantly in the p-T-miR-7 -transfected group (p < 0.05). [score:1]
Further analysis showed that miR-7 could bind to the 3′ UTR region of NDUFA4 mRNA (Figure 4G). [score:1]
It was noticed that none of these biochemistry indicators changed significantly (Figure 7C; p > 0.05), indicating that there were not any changes on biological function of important organs and tissues in the p-T-miR-7 injection group. [score:1]
17, 18 Therefore, we further investigated whether expression of miR-7 operated by TTF-1 promoter could affect the growth of human lung cancer cells in vitro. [score:1]
95D cells were seeded in 96-well plates at 1 × 10 [4]/well with triplicate and transiently transfected with p-T-miR-7 plasmid (2 μg), p-NDUFA4 plasmid (10 μg), or p-Cont plasmid (2 μg/10 μg). [score:1]
MicroRNA-7 (miR-7), a unique member of miRNAs, played an important role in the progression of various tumors including lung cancer. [score:1]
As shown in Figure S2, the copies of the p-T-miR-7 plasmid was higher in lung tissue and tumor tissue (p < 0.05), but not in the other organs and tissues, indicating the plasmid was mainly distributed in the lung tissue and tumor tissue in vivo. [score:1]
As shown in Figure 7A, there were no significant changes in the morphology of six important organs and tissues, including heart, liver, spleen, kidney, brain, and lymph nodes, between the p-T-miR-7 injection group and the control group (p > 0.05). [score:1]
95D cells were transiently transfected with p-T-miR-7 plasmid (2 μg), p-NDUFA4 plasmid (10 μg), or p-Cont plasmid (2 μg/10 μg), as described above. [score:1]
As shown in Figure 2H, H&E staining showed that there were large areas of necrosis in tumor tissue in the p-T-miR-7 injection group. [score:1]
Human lung cancer 95D cells were transiently transfected with p-T-miR-7 plasmid (2 μg), p-NDUFA4 plasmid (10 μg), or p-Cont plasmid (2 μg/10 μg) or scramble control as described above. [score:1]
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Overlay of the miR-7 predicted target genes with down-regulated genes with a functional annotation in neovascularization led to selection of OGT as putative novel target of miR-7. OGT is an enzyme that is involved in the hexosamine biosynthetic pathway which adds an O-GlcNAc moiety to the free hydroxyl group of select serine and threonine residues on a diverse population of nuclear and cytosolic proteins [35, 36]. [score:8]
Both siRNA against OGT and Alloxan did not inhibit EC migration or EC tube formation in vitro, suggesting that OGT is a good marker gene of miR-7, but not the prime miR-7 target through which EC cell migration and tube formation are inhibited (Supplementary Fig. S3 and S4). [score:7]
The majority, 1317, of the miR-7 modulated genes were down-regulated while 1183 were upregulated. [score:7]
These data suggest that inhibition of angiogenesis is the prime mechanism for the N2A tumor growth suppression upon intratumoral delivery of miR-7. The lack of efficacy on tumor cell proliferation in vivo is corroborated by the observation that miR-7 did not inhibit cell viability of N2A cells in vitro (Supplementary Fig. S5). [score:7]
Furthermore, not only tumor vessel density was reduced, expression of the nuclear proliferation marker Ki-67 and expression of the newly identified miR-7 target gene OGT were also affected by miR-7 treatment in vivo. [score:7]
Differentially expressed genes that are regulated by miR-7 and are a predicted target genes of miR-7 are plotted. [score:6]
Similar to EC, downregulation of OGT in U-87 MG cells by RNAi interference did not inhibit cell viability (Supplementary Fig. S9), indicating that also in tumor cells OGT is a good marker for miR-7 delivery but not for miR-7 efficacy. [score:6]
First, the set of regulated genes was overlaid with the 1051 predicted miR-7 targets, based on the TargetScan prediction tool in IPA. [score:6]
It is noted that other miR-7 targets, such as EGFR and PI3K were not down-regulated in miR-7 treated EC based on the RNA-seq analysis (Supplementary Table S7), suggesting that the anti-proliferative activity of miR-7 occurs through other pathways in EC than in tumor cells. [score:6]
In total 282 predicted miR-7 targets were up- or downregulated within the set of 2500 genes (Fig. 2b). [score:6]
Here we show for the first time that OGT is a target of miR-7, suggesting that the anti-angiogenic effect of miR-7 in EC can, at least partly, be mediated by downregulation of OGT. [score:6]
Anti-angiogenic activity of miR-7 mimic in vivo– systemic tumor neovasculature targetingClinical application of miRNA -based therapeutics is dependent on systemic administration and intracellular delivery of the miRNA (mimic) to the target site. [score:5]
Investigation of gene expression levels of known miR-7 target shows that EGFR and PI3K and most of the other known miR-7 targets are not affected in HUVEC after miR-7 transfection (Supplementary Fig S10). [score:5]
Eight out of 11 genes that are associated to novel anti-angiogenic drugs are down regulated Genes highly involved in tumor angiogenesis Expression ratio after miR-7 treatment versus control (log2(ratio)) Example of drug in clinical trial VEGF-B 1.443 Aflibercept (Regeneron) VEGF-C −0.778 VGX-100 (Circadian) Angiopoeitin-2 −0.805 PF-04856884 (Pfizer) PDGF-D 0.899 CR-002 (CuraGen) Jagged-1 −0.739 RO4929097 (Roche) ADAM-10 −0.870 INCB3619 (InCyte) FGF-2 −1.732 Gal-F2 (Galaxy/Roche) CXCR4 1.169 BMS-936564 (Bristoll Myers Squibb) S1PR1 −1.108 Fingolimod (Novartis) S1PR3 −1.035 Fingolimod (Novartis) NRP1 −0.801 MNRP1685A (Genentech) antibodyIPA was also used to identify biological processes that are regulated by miR-7 (Supplementary Table S3). [score:5]
Differential expression analysis showed that 2500 genes were significantly up- or downregulated after transfection of HUVEC with miR-7 mimic (p-value < 0.05, compared to miR-Scr, Fig. 2a). [score:5]
However, inhibition of OGT in EC does not inhibit EC tube formation or migration, which indicates that OGT is a suitable biomarker for miR-7 activity, but does not explain the anti-angiogenic activity of miR-7 in EC. [score:5]
The dual targeting of the cRGD-coated nanoparticles to both endothelial as well as cancer cells provides potential for high therapeutic efficacy of miR-7. Together with the well-known role of miR-7 as regulator of invasion and migration in cancer, the novel anti-angiogenic property of miR-7 and its regulation of diverse genes involved in angiogenesis strengthen its potential value as therapeutic agent for the treatment of cancer Human Umbilical Vein Endothelial Cells (HUVEC) (Lonza) were cultured in EBM-2 medium (Lonza) supplemented with bullet kit (EGM-2, Lonza) containing several growth factors and 10% Fetal Calf Serum (FCS) (Sigma). [score:5]
Eight out of 11 genes that are associated to novel anti-angiogenic drugs are down regulated Genes highly involved in tumor angiogenesis Expression ratio after miR-7 treatment versus control (log2(ratio)) Example of drug in clinical trial VEGF-B 1.443 Aflibercept (Regeneron) VEGF-C −0.778 VGX-100 (Circadian) Angiopoeitin-2 −0.805 PF-04856884 (Pfizer) PDGF-D 0.899 CR-002 (CuraGen) Jagged-1 −0.739 RO4929097 (Roche) ADAM-10 −0.870 INCB3619 (InCyte) FGF-2 −1.732 Gal-F2 (Galaxy/Roche) CXCR4 1.169 BMS-936564 (Bristoll Myers Squibb) S1PR1 −1.108 Fingolimod (Novartis) S1PR3 −1.035 Fingolimod (Novartis) NRP1 −0.801 MNRP1685A (Genentech) antibody IPA was also used to identify biological processes that are regulated by miR-7 (Supplementary Table S3). [score:5]
The results demonstrate that targeted systemic delivery of miR-7 inhibited tumor angiogenesis and growth. [score:5]
miR-7 mediated differential expression of genes that are currently clinically explored as anti-angiogenic drug targets. [score:5]
After transfection of HUVEC with miR-7 mimic cell proliferation was inhibited in a concentration dependent manner, up to 50% inhibition compared to a negative control miRNA with a scrambled sequence (miR-Scr). [score:4]
Indirect evidence for systemic delivery of miR-7 to the tumor tissue was provided by the observation that mice treated with the miR-7 mimic -loaded, integrin -targeted nanoparticles had smaller, pale and less vascularized tumors than control mice. [score:4]
Not only did miR-7 inhibit growth factor induced angiogenesis in vitro but miR-7 also impaired developmental angiogenesis in chicken embryo CAM with potency comparable to sunitinib. [score:4]
MiR-7 not only inhibited EC proliferation by 50%, but also inhibited migration of EC by nearly 70%. [score:4]
Downregulation of OGT by miR-7 is not EC specific, but was also confirmed in U-87 MG cells in vitro (Supplementary Fig. S8). [score:4]
In earlier studies, miR-7 was not picked up as miRNA involved in angiogenesis in an EC specific differential expression screen (13), which may suggest that miR-7 has no physiological role in EC to regulate angiogenesis. [score:4]
The observed downregulation of OGT in miR-7 transfected HUVEC was confirmed by RT-PCR and Western Blot analysis over a concentration range of 15-100 nM (Fig. 2c and d). [score:4]
Overlay of strongly down-regulated angiogenesis -associated genes (log2 ratio (miR-7 vs. [score:4]
Figure 4(a) miR-7 inhibits tumor growth after local delivery. [score:3]
miR-Scr), p-value <0.05) with predicted miR-7 targets (Supplementary Table S4) pointed to O-linked β-N-acetylglucosamine transferase (OGT) as potentially important mediator of miR-7 action. [score:3]
Together, these studies demonstrate strong anti-angiogenic activity of miR-7 upon overexpression in EC. [score:3]
U-87 MG tumor bearing mice were treated with miR-7 mimic using a cyclic Arginine-Glycine-Aspartic acid (cRGD) peptide coupled biodegradable polyamide nanoparticles, targeting integrins αvβ3 and αvβ5. [score:3]
The observed inhibition of tumor growth can thus be ascribed to a combination of an anti-angiogenic effect of miR-7 delivered to tumor -associated EC and an anti-proliferative effect of miR-7 delivered to tumor cells. [score:3]
The log2(ratio) was calculated as described in Material and Methods and reflects the differential expression of genes in HUVEC treated with miR-7 and gene expression in HUVEC treated with miR-Scr. [score:3]
The U-87 MG xenograft tumor mo del was selected because it is highly vascularized and responds well to anti-angiogenic therapy such as bevacizumab or sunitinib [40, 41], making it a suitable mo del to study the anti-angiogenic property of miR-7. Naturally, this mo del does not reflect the nature of glioblastomas in the clinic and recently it was shown that angiogenesis inhibition in glioblastoma patients may induce glioblastoma migration and invasion [42, 43]. [score:3]
To explain the anti-angiogenic properties of miR-7, gene expression analysis was performed using RNA-seq. [score:3]
Indeed, miR-7 inhibited wound closure while miR-Scr transfected cells did not (Fig. 1h). [score:3]
This is indicative of a strong anti-angiogenic activity of miR-7. This was supported by the observation that treatment of CAM with a clinically approved multikinase anti-angiogenic drug, sunitinib, showed a similar inhibitory effect on vascularization. [score:3]
Athymic Nude-Foxn1 [nu] mice bearing U-87 MG tumors were injected intravenously with αvβ3/αvβ5 targeted miR-7 nanoparticles (3 mg/kg miRNA). [score:3]
Indeed, miR-7 inhibits proliferation of not only HUVEC, but also of U-87 MG cells in vitro. [score:3]
Anti-angiogenic activity of miR-7 mimic in vivo– systemic tumor neovasculature targeting. [score:3]
This suggests that inhibition of miR-7 mediated tumor growth is caused by a combined effect on both tumor cells and tumor EC. [score:3]
miR-7 modifies endothelial gene expression. [score:3]
To confirm effective delivery of miR-7 into the tumor tissue we performed IHC staining of OGT, one of the target genes of miR-7 (Fig. 5g). [score:3]
Inhibitory effect of miR-7 on tumor growth by local delivery. [score:3]
In a functional screen with a lentiviral miRNA library miR-7 was identified as inhibitor of EC proliferation. [score:3]
Figure 2(a) miR-7 changes the expression of 2500 HUVEC genes after transfection. [score:3]
Our observation that miR-7 inhibits proliferation of U-87 MG cells confirms the anti-tumorigenic effect of miR-7 in tumor cells. [score:3]
Delivery of miR-7 using this novel formulation demonstrated inhibition of tumor growth in a human glioblastoma xenograft mo del. [score:3]
Inhibitory effect of miR-7 on tumor growth by systemic delivery. [score:3]
Figure 1Anti-angiogenic property of miR-7 in vitro(a) miR-7 inhibits HUVEC cell viability. [score:3]
The decrease in luciferase activity in the presence of miR-7 indicates a direct interaction between miR-7 and the 3′UTR of OGT (Fig. 2e). [score:2]
This study has identified miR-7 as a prominent regulator of angiogenesis. [score:2]
The mechanism through which miR-7 regulates distinct pathways in EC and tumor cells will be subject of future research. [score:2]
To proof that OGT is a direct target gene of miR-7, we measured luciferase activity in Hela cells transfected with a plasmid containing the 3′UTR sequence of OGT. [score:2]
Eight out of 11 genes that are associated to novel anti-angiogenic drugs are down regulated Mature miR-7 is conserved between chicken, human, and mouse (Fig. 3a). [score:2]
In more complex in vitro angiogenesis assays, miR-7 inhibited the ability of HUVEC to form two-dimensional tubules on matrigel and three-dimensional sprouts in collagen. [score:2]
Tumors of the miR-7 treatment group were pale and less vascularized compared to those in control groups (Fig. 5a) and systemic delivery of miR-7 mimic inhibited tumor growth by 42% after two weeks of treatment (Fig. 5b). [score:2]
miR-7 treated mice showed significant tumor growth inhibition compared to vehicle treated mice. [score:2]
Caspase-3 cleavage in HUVEC treated with miR-7 suggests that part of the anti-proliferative activity results in cell apoptosis (Supplementary Fig. S2b). [score:1]
Tumor treatment with intratumoral injection of miRNA delivered by electroporation showed that the miR-7 mimic decreased angiogenesis and reduced tumor growth. [score:1]
Therefore, this approach was selected for systemic delivery of miR-7. Hereto, a novel biodegradable neovasculature targeted nanoparticles formulation was developed and used to investigate the anti-tumor activity of miR-7 following intravenous administration in human glioblastoma U-87 MG bearing mice. [score:1]
Systemic delivery of miR-7 nanoparticles in tumor bearing mice was a randomized, blinded study which was performed in an AALAC certified vivarium (Biomedical Research Institute, Rockville, MD, USA). [score:1]
Gene modulation by miR-7 mimic – HUVEC culture. [score:1]
Stem-Loop RT-PCR showed that the pre-miRNA-7 hairpin is processed into mature miR-7 (hsa-miR-7-5p, Supplementary Table S2). [score:1]
The anti-proliferative effect of miR-7 was comparable to that of a positive control, i. e. siRNA against Polo-like kinase 1 (Plk1), a cell proliferation kinase. [score:1]
Cells were transfected with increasing concentrations of miR-7, miR-scr or siPLK-1. siPLK-1 and miR-scr were used as positive and negative control. [score:1]
Stem-loop RT-PCR was used to determine the relative tumor amounts of miR-7 in the different treatment groups. [score:1]
The anti-angiogenic property of miR-7 was also observed microscopically with a statistically significant reduction of immunohistochemical staining of CD31 in tumor tissue from the miR-7 treated mice (Fig. 5c and d). [score:1]
Hela cells seeded in 24-well plated, were co -transfected with 10 nM miR-7 or miR-Scr and 100ng/well 3′UTR psiCHECK [TM]-2 construct using Lipofectamine 2000 (Invitrogen) according to manufacturers protocol. [score:1]
This was confirmed by in vitro studies that demonstrated reduced proliferation of U-87 MG cells upon transfection with miR-7 mimic (Supplementary Fig. S7). [score:1]
Moreover, tumor tissue from the miR-7 treated mice contained considerable amounts of necrotic lesions, which is an indication of hypoxia from reduced angiogenesis. [score:1]
We developed a novel αvβ3/αvβ5-integrin targeted nanoparticles for systemic delivery of miR-7 mimic to tumor EC and tumor cells and evaluated it in a human glioblastoma xenograft tumor mo del. [score:1]
Glioblastoma xenograft tumor in vivo mo del: systemic deliverySystemic delivery of miR-7 nanoparticles in tumor bearing mice was a randomized, blinded study which was performed in an AALAC certified vivarium (Biomedical Research Institute, Rockville, MD, USA). [score:1]
Chick CAMs were treated locally within a nitrocellulose ring with 300 picomol miR-7 or miR-Scr using Lipofectamine 2000 or with 200 picomol sunitinib. [score:1]
Mutagenesis of the 3′UTR sequence of the predicted binding site of miR-7 (Fig. 2f) restored luciferase activity, thereby confirming the specificity of the interaction between miR-7 and the OGT 3′UTR (Fig. 2e). [score:1]
Anti-angiogenic activity of miR-7 mimic in vitro. [score:1]
The effect of miR-7 on tumor cell proliferation was determined by Ki-67 staining (in brown). [score:1]
AJ mice bearing tumors with Neuro2A cells were treated locally with 10 μg miR-7 or 10 μg miR-Scr or PBS by intratumoral injection and electroporation. [score:1]
The biochemical process underlying tumor growth inhibition by miR-7 mimics was investigated using immunohistochemical (IHC) detection of CD31, an endothelial cell marker for microvessel density (Fig. 4c). [score:1]
Figure 3(a) Seed sequence of miR-7. Illustration of conserved seed sequence of miR-7 among different species. [score:1]
Anti-angiogenic property of miR-7 in vitro. [score:1]
HUVEC, seeded in a 6-well plate (8×10 [4] cells/well), were transfected at different concentrations of miR-7 using X-tremeGENE (see above). [score:1]
Systemic delivery of miR-7 mimic not only reduced tumor angiogenesis but also reduced tumor proliferation as demonstrated by the statistically significant reduction in Ki-67 staining of miR-7 treated tumor tissue (Fig. 5e and f). [score:1]
HUVEC were transfected with 50 nM miR-7 or miR-scr. [score:1]
Subsequently, the rings were loaded with 300 picomol miR-Scr or miR-7 mimics complexed with Lipofectamine 2000 in 20 mM Hepes buffered glucose (pH 7,4). [score:1]
Apart from the here described newly discovered anti-angiogenic effect, human miR-7 was first described by Lim et al. in 2003 [28] and later this miRNA was found to be associated with anti-tumorigenic effects in glioma, hepatocellular carcinoma cells, and head and neck cancer cells [29- 34]. [score:1]
In this work, the U-87MG mo del was used to explore the feasibility of miR-7 as anti-angiogenic agent and other therapeutically relevant tumor mo dels will be explored in the near future. [score:1]
The most potent miRNA, miR-7, was validated for anti-angiogenic activity in vitro. [score:1]
miR-7 treated mice showed a statistically significant reduction in OGT levels (Fig. 5h). [score:1]
Anti-angiogenic activity of miR-7 mimic in vivo– local tumor administration. [score:1]
A reduction in vascular density in the regions between large blood vessels was visible in CAM treated with miR-7 mimic while vascular density was not reduced in untreated or miR-Scr treated CAM (Fig. 3b). [score:1]
Relative expression was calculated as the ratio of reads mapping to a gene in the miR-7 transfected sample and the reads mapping to a gene in the miR-Scr transfected sample. [score:1]
Figure 5(a) miR-7 treated animals show pale and less vascularized tumors. [score:1]
Delivery of miR-7 by electroporation into the tumor tissue was determined by stem loop RT-PCR. [score:1]
HUVEC were transfected with increasing concentrations of miR-7 or miR-Scr. [score:1]
We therefore selected miR-7 for further validation as an anti-angiogenic miRNA candidate. [score:1]
Anti-proliferative effect of systemically delivered miR-7 was determined by Ki-67 staining, indicated as brown spots. [score:1]
HUVEC were transfected with increasing concentration of miR-7 or miR-Scr. [score:1]
HUVEC were transfected with either miR-7 or miR-Scr and the transcriptome was quantified by RNA-Seq. [score:1]
To elucidate the mechanism of action by which miR-7 exerts the anti-angiogenic effects, transcriptional analysis of miR-7 mimic transfected HUVEC was performed. [score:1]
HUVEC were transfected with 50 nM miR-7 or miR-scr and seeded on matrigel at 48hrs after transfection. [score:1]
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To identify the putative miR-7, miR-34a, and miR-504 targets in the Shank3 interactome, we first pooled all the putative targets for each miRNA from six different prediction tools (TargetScan [http://targetscan. [score:9]
We found that miR-7, miR-34a, and miR-504, three miRNAs with altered expression profiles in multiple neuropsychiatric disorders, directly regulate SHANK3 expression. [score:7]
To understand whether Shank3 expression was required for the spine changes observed after miR-7 or miR-504 inhibition, we co -transfected a previously validated siRNA targeting mouse Shank3 [12]. [score:7]
Expression of miR-7 decreased the luciferase activity of the wild-type SHANK3 3′UTR construct in HEK293T cells, indicating that miR-7 inhibited its expression (Fig.   1b). [score:7]
miR-7, miR-34a, and miR-504 directly regulate the expression of SHANK3Using the TargetScan prediction tool (Release 6.2, http://www. [score:7]
As miR-7, miR-34a, and miR-504 are expressed in mouse hippocampal neurons [39– 41], we decided to test the effect of inhibition of endogenous miRNAs on SHANK3 expression and dendritic spines. [score:7]
Together, these results suggest that miR-7, miR-34a, and miR-504 directly bind to the SHANK3 3′UTR and downregulate its expression. [score:7]
Transfection of locked-nucleic acid (LNA) inhibitors against each of the miRNAs increased the luciferase activity of wild-type, but not the respective binding site-mutant, SHANK3 3′UTR constructs, suggesting that endogenous miR-7, miR-34a, and miR-504 could regulate SHANK3 expression (Fig.   3f and g). [score:6]
Furthermore, the expression of rat Shank3 3′UTR, where the second miR-7 binding site of human 3′UTR is not conserved, was still reduced by miR-7 in the luciferase assay (Fig.   1b), indicating that the first site in the SHANK3 3′UTR is the only functional target site for miR-7. Expression of miR-34a or miR-504 also decreased the luciferase activity of the wild-type SHANK3 3′UTR construct in HEK293T cells (Fig.   1c and d). [score:6]
This circular RNA can indirectly increase the expression of other miR-7 targets by sequestering miR-7, and was thus named as a circular RNA sponge for miR-7 (ciRS-7) [42]. [score:6]
Together, our results provide new insight into the miRNA -mediated regulation of SHANK3 expression, and its potential implications for multiple neuropsychiatric disorders with altered expression profiles of miR-7, miR-34a, and miR-504. [score:6]
Indeed, in HEK293T cells, ciRS-7 expression partially blocked the inhibitory effect of miR-7 on the luciferase activity of the wild-type, but not the miR-7 binding-mutant SHANK3 3′UTR construct (Fig.   3j). [score:5]
g LNA -inhibitor against miR-7, miR-34a, and miR-504 did not affect the expression of the mutant SHANK3 3′UTR in cultured neurons. [score:5]
These results suggest that miR-7, miR-34a, and miR-504 could also potentially regulate the expression of some Shank3-interacting proteins, especially those involved in actin regulation. [score:5]
Nevertheless, after normalization with EGFP, the expression of Shank3 with 3′UTR, but not that without 3′UTR, was significantly decreased by miR-7. e Co-transfection of the Shank3 construct without the 3′UTR, but not that with the 3′UTR, rescued the decreased dendritic spine density by miR-7 or miR-504 overexpression (n = 20–30). [score:5]
miR-7, miR-34a, and miR-504 directly regulate the expression of SHANK3. [score:5]
f LNA -inhibitor against miR-7, miR-34a, or miR-504 increased the expression of co -transfected SHANK3 3′UTR in cultured mouse hippocampal neurons. [score:5]
When we picked those targets suggested by more than two prediction tools, 79, 67, and 46 genes in the Shank3 interactome were revealed as putative targets for miR-7, miR-34a, and miR-504, respectively (Fig.   2a). [score:5]
Moreover, opposite to the miRNA overexpression, miR-7 or miR-504 inhibition increased spine density in cultured mouse hippocampal neurons (Fig.   3h). [score:5]
Moreover, overexpression or inhibition of miR-7 and miR-504 affected the dendritic spines of the cultured hippocampal neurons in a Shank3 -dependent manner. [score:5]
Taken together, these results suggest that miR-7, miR-34a, and miR-504 could regulate SHANK3 expression in cultured mouse hippocampal neurons, and that miR-7 and miR-504 could regulate dendritic spines in a Shank3 -dependent manner. [score:5]
d Representative western blot images show that overexpression of miR-7 or miR-504 decreased the expression of the rat Shank3 construct with the 3′UTR, but not of that without the 3′UTR, in HEK293T cells. [score:5]
a Summary of the process identifying putative miR-7, miR-34a, and miR-504 targets in the Shank3 interactome (top), and the GO analysis of the putative targets (bottom). [score:5]
In this study, we examine this possibility for the SHANK3 gene and report post-transcriptional regulation of SHANK3 expression by three miRNAs, miR-7, miR-34a, and miR-504, which were previously shown to be altered in some neuropsychiatric disorders that could also be caused by SHANK3 dosage changes. [score:4]
In this study, we showed post-transcriptional regulation of SHANK3 expression in neurons by three miRNAs, miR-7, miR-34a, and miR-504. [score:4]
d Mutation of the miR-7 binding site blocked its repressive effect on the expression of the human PFN2 3′UTR. [score:4]
We first pooled the putative human target genes for each of the three miRNAs from six prediction tools (total 8,246 targets for miR-7, 4,635 for miR-34a, and 3,392 for miR-504), and then compared these lists with 338 Shank3 interactome genes [12]. [score:4]
Together, these results suggest that miR-7, miR-34a, and miR-504 could commonly regulate the expression of at least six major Shank3 isoforms (a, a[E10-12S V], c, d, e, and f) (Additional file 1: Figure S3) [8, 49]. [score:4]
Importantly, these dendritic spine changes were dependent on Shank3, suggesting that Shank3 functions as a core protein among the actin-related miR-7 and miR-504 targets in regulating dendritic spines. [score:4]
We further mutated the putative miRNA binding sites in the 3′UTRs of PFN2 and SPTBN2, the two genes most downregulated by miR-7 and miR-504, respectively, and found that the mutations abolished the effect of the miRNAs in the luciferase assays (Fig.   2d and e). [score:4]
Here we show post-transcriptional regulation of SHANK3 expression by three microRNAs (miRNAs), miR-7, miR-34a, and miR-504. [score:4]
miR-7, miR-34a, and miR-504 potentially regulate other targets in the Shank3 interactome. [score:4]
Altered expression profiles of miR-7, miR-34a, and miR-504 in multiple neuropsychiatric disorders. [score:3]
c Overexpression of miR-7 or miR-504 decreased the density of dendritic spines in cultured mouse hippocampal neurons (n = 16–30). [score:3]
However, only the Shank3 construct without the 3′UTR could rescue the decreased spine density in response to miR-7 or miR-504 overexpression (Fig.   3e). [score:3]
The expression of Shank3 construct with 3′UTR, but not that without 3′UTR, was significantly decreased by miR-7 and miR-504 in mouse cultured neurons. [score:3]
We transfected enhanced green fluorescent protein (EGFP) -expressing plasmid with control miRNA, miR-7, or miR-504 into cultured mouse hippocampal neurons, and immunostained the neurons with GFP antibody to visualize dendritic protrusions. [score:3]
TargetScan predicted that the SHANK2 3′UTR contained conserved miR-7, but not miR-34a or miR-504, binding sites. [score:3]
g miR-7, miR-34a, and miR-504 synergistically decreased the expression of the SHANK3 3′UTR. [score:3]
Recently, Zhang et al. claimed that the miR-7/ SHANK3 axis could be involved in schizophrenia pathogenesis, showing an inverse correlation between the expression levels of miR-7 and SHANK3 [35]. [score:3]
i Co-transfection of Shank3 siRNA blocked the increase of dendritic spines in response to miR-7 or miR-504 inhibition (n = 16–20). [score:3]
Indeed, in HEK293T cells, miR-7 and miR-504 reduced the expression of Shank3 proteins from the construct with the 3′UTR, but did not affect the construct without the 3′UTR (Fig.   3d). [score:3]
Statistical analyses are in 1: Table S3The validated target sites for miR-7, miR-34a, and miR-504 were highly conserved across different species (Fig.   1e). [score:3]
i miR-7 did not affect the expression of the human SHANK2 3′UTR that contains two putative miR-7 binding sites. [score:3]
h LNA -inhibitor against miR-7 or miR-504 increased dendritic spines in cultured neurons (n = 18–20). [score:3]
The expression of miR-7, miR-34a and miR-504 were reported to be altered in the postmortem brains, fibroblasts, or blood samples of patients with schizophrenia, depression, or bipolar disorder (Additional file 1: Table S2) [29– 34]. [score:3]
a miR-7, miR-34a, or miR-504 overexpression decreased the luciferase activity of wild-type SHANK3 3′UTR in cultured mouse hippocampal neurons. [score:3]
When expressed at a fixed total amount, miR-7, miR-34a, and miR-504 in combination decreased the luciferase activity of SHANK3 3′UTR more efficiently than each miRNA alone, suggesting their synergistic effect (Fig.   1g). [score:3]
Neither miR-7 nor ciRS-7 changed the expression of miR-7 binding-mutant SHANK3 3′UTR. [score:3]
Notably, we observed decreased expression of EGFP when miR-7 was cotransfected. [score:3]
Statistical analyses are in 1: Table S3 The validated target sites for miR-7, miR-34a, and miR-504 were highly conserved across different species (Fig.   1e). [score:3]
We found that both miR-7 and miR-504 overexpression decreased dendritic spine density, but neither of the miRNAs affected filopodia (Fig.   3c). [score:3]
As ciRS-7 and miR-7 were detected in neuronal tissues including the hippocampus [42, 43], we examined the effect of ciRS-7 overexpression on dendritic spines. [score:3]
j ciRS-7 partially blocked the repressive effect of miR-7 on the expression of wild-type SHANK3 3′UTR in HEK293T cells. [score:3]
miR-7 and miR-504 regulate dendritic spines of cultured hippocampal neurons. [score:2]
However, miR-7 did not change the expression of SHANK2 3′UTR in luciferase assays (Fig.   1i), which might be, at least partly, due to the difference in the secondary structures of SHANK3 and SHANK2 3′UTRs (Additional file 1: Figure S1). [score:2]
Fig. 2Regulation of Shank3-interacting actin-related proteins by miR-7, miR-34a, and miR-504. [score:2]
Therefore, we searched for proteins in the Shank3 interactome that might also be regulated by miR-7, miR-34a, or miR-504. [score:2]
These lists (8,246 targets for miR-7, 4,635 for miR-34a, and 3,392 for miR-504) were compared with the 388 proteins in the Shank3 interactome. [score:2]
c miR-7, miR-34a, and miR-504 decreased the expression of the 3′UTRs of some Shank3-interacting proteins in luciferase assays. [score:2]
However, neither the direct binding of miR-7 to the SHANK3 3′UTR nor its functional effect on neuronal synapses have been reported. [score:2]
Between the two putative miR-7 binding sites in the SHANK3 3′UTR (523–530 and 1, 080–1, 086), mutation of the first (523–530) abolished the effect of miR-7 on luciferase activity (Fig.   1b). [score:2]
We found that cultured hippocampal neurons transfected with ciRS-7 showed increased spine density compared to the neurons transfected with control plasmid (ciRS-7-ir) (Fig.   3k), which was the same phenotype as that observed in response to a miR-7 LNA inhibitor (Fig.   3h). [score:2]
We found that Shank3 siRNA alone resulted in lower spine density compared to that observed with control siRNA, and that it also blocked the increase in spine density in response to miR-7 or miR-504 inhibition (Fig.   3i). [score:2]
h miR-7, miR-34a, and miR-504 did not affect the mRNA levels of the SHANK3 3′UTR. [score:1]
f The sequence alignments across species for the miR-7 and miR-34a binding sites in the PFN2 and SPTBN2 3′UTRs, respectively. [score:1]
b miR-7, miR-34a, and miR-504 did not affect the luciferase activity of the mutant SHANK3 3′UTR constructs in cultured neurons. [score:1]
Consistent with the results from HEK293T cells (Fig.   1), miR-7, miR-34a, and miR-504 decreased the luciferase activity of the wild-type, but not the respective binding site-mutant, SHANK3 3′UTR constructs (Fig.   3a and b). [score:1]
The luciferase activity of the rat Shank3 3′UTR (rWT), containing only the first binding site, was still reduced by miR-7. RL, Renilla luciferase; FL, firefly luciferase. [score:1]
We transfected cultured hippocampal neurons with control miRNA, miR-7, or miR-504 in combination with the two Shank3 constructs. [score:1]
b miR-7 decreased the luciferase activity of the wild-type (hWT), but not the first binding site-mutant (hM), SHANK3 3′UTR. [score:1]
To test the effect of a circular RNA sponge for miR-7, 200 ng of ciRS-7-ir or ciRS-7 plasmids (kindly gifted from Dr. [score:1]
Consistently, the expression of Shank3 construct with 3′UTR, but not that without 3′UTR, was decreased by miR-7 and miR-504 in cultured neurons measured by immunostaining (Additional file 1: Figure S2). [score:1]
Jorgen Kjems) were co -transfected with 30 ng of psiCHECK-2 SHANK3 3′UTR and 6 pmol of miR-7 duplex. [score:1]
Recently, an endogenous circular RNA that has more than 70 binding sites for miR-7 was identified [42, 43]. [score:1]
Finally, we chose three miRNAs, miR-7, miR-34a, and miR-504 because of their strong 8-mer type binding sites [20] in the SHANK3 3′UTR. [score:1]
Consistently, both gain- and loss-of-function experiments showed that miR-7 and miR-504 control actin-rich dendritic spines. [score:1]
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Nine of these deregulated miRNAs were confirmed to be either up-regulated (2 miRNAs) or down-regulated (7 miRNAs) with miR-7 being the most significantly down-regulated one (8.9-fold, p < 0.01) in 3 independent assays (Figure 1C). [score:10]
miR-7 suppresses the expressions of EGFR, IGF-1R and CRAF that are up-regulated in VemR melanoma cells. [score:8]
miR-7 suppresses the expressions of EGFR, IGF-1R and CRAF that are up-regulated in VemR A375 cells. [score:8]
Since PTEN also plays an important role in tumor cell migration and spreading, we speculated that miR-7 could not only reverse the resistance of BRAF inhibitors, but also suppress the invasion and migration of VemR A375 cells through up -regulating the expression of PTEN. [score:8]
To further identify the target genes of miR-7 and investigate how miR-7 regulates the expression of these targets to reverse BRAFi resistance, we first used TargetScan (http://www. [score:8]
TargetScan and PicTar anaysis also predicted that PTEN could be a potential target gene of miR-7. Our additional study further demonstrated that the expression level of PTEN mRNA was significantly increased after transfection with miR-7 for 48 hrs (Supplementary Figure S5E). [score:7]
These data also suggested that miR-7 could inhibit VemR melanoma cell proliferation through combined inhibition of EGFR/IGF-1R/CRAF expressions, which further decreased the levels of p-ERK and p-AKT in VemR A375 cells. [score:7]
Therefore, our study indicated that miR-7 could regulate vemurafenib resistance by suppressing the over -expression of EGFR, IGF-1R and CRAF in VemR A375 cells. [score:6]
The expression level of miR-7 was also down-regulated in Mel-CVR cells. [score:6]
After transfection with miR-7 mimics for 72 hrs, the expression level of miR-7 in Mel-CVR cells was significantly up-regulated (p < 0.05) (Supplementary Figure S1C). [score:6]
Seventeen miRNAs were found which had 2-folds or greater differences in levels in VemR A375 melanoma cells as compared with parental A375 cells by microarray (Figure 1B and Supplementary Table S1), with 7 down-regulated miRNAs including miR-7 (40.3-fold), miR-18a-5p (5.2-fold), miR-19a-3p (3.6-fold), miR-20b-5p (3.4-fold), miR-17-5p (3.2-fold), miR-20a-5p (3.1-fold), and miR-19b-3p (2.8-fold) and 10 up-regulated miRNAs including miR-514a-3p (116-fold), miR-129-1-3p (87-fold), miR-509-3p (83-fold), miR-629-3p (22-fold), miR-937-5p (4.6-fold), miR-3960 (4.3-fold), miR-1915-3p (3.2-fold), miR-6090 (3.1-fold), miR-4281 (2.6-fold) and miR-4634 (2-fold). [score:6]
miR-7 is down-regulated in VemR A375 and Mel-CVR melanoma cells and reestablishment of miR-7 expression reverses the resistance to vemurafenib. [score:6]
miR-7 is down-regulated in VemR A375 melanoma cells and reestablishment of miR-7 expression sensitizes VemR A375 melanoma cells to vemurafenib. [score:6]
Some miR-7 associated cancer research have also shown that miR-7 could attenuate the activation of PI3K/AKT and MAPK signaling pathways [32- 41], leading to suppression of tumor cell proliferation/survival and inhibition of tumor invasion/metastasis. [score:5]
Our studies also demonstrated that reestablishment of miR-7 expression could reverse the resistance to vemurafenib and inhibited resistant A375 melanoma cell growth both in vitro and in xenograft tumor mo dels. [score:5]
In addition of its decreased expression in VemR A375 cells, miR-7 was also identified to be down-regulated in Mel-CVR melanoma cells as compared with parental Mel-CV cells by analysis (p < 0.05) (Supplementary Figure S1B). [score:5]
These results indicated that miR-7 was associated with vemurafenib resistance and reestablishment of miR-7 expression was capable of inhibiting the proliferation of both VemR A375 and Mel-CVR cells and reversing the resistance to vemurafenib. [score:5]
miR-7 could decrease the expressions of EGFR, IGF-1R and CRAF and further suppressed the activation of MAPK and PI3K/AKT pathways in VemR A375 melanoma cells. [score:5]
Our studies indicated that miR-7 could decrease the expression of EGFR, IGF-1R and CRAF and partly reverse the resistance to BRAFi in VemR melanoma cells, further suppressed the tumor growth in VemR melanoma xenografted mice mo del. [score:5]
Furthermore, by decreasing the expression levels of EGFR, IGF-1R and CRAF, miR-7 could inhibit the activation of RAS/RAF/MEK/ERK (MAPK) and PI3K/AKT pathway and partially reverse the resistance to BRAFi in VemR A375 melanoma cells. [score:5]
Introduction of miR-7 could decrease the expression levels of EGFR, IGF-1R and CRAF in vitro as well as in VemR A375 melanoma xenograft mice mo dels, which indicated that EGFR, IGF-1R and CRAF were the target genes of miR-7 that are closely associated with the acquired resistance to BRAFi in VemR melanoma. [score:5]
In summary, our results demonstrated for the first time that miR-7 expression was decreased in both VemR A375 and Mel-CVR melanoma cells and its low expression contributed to BRAFi resistance. [score:5]
Compared with miRNA -negative control (miR-NC), VemR A375 cell viability was reduced by 30% (p < 0.05) only in miR-7 mimics transfection group while the other six down-regulated miRNA mimics did not show any significant inhibitory effects on VemR cell proliferation (Figure 1D). [score:5]
However, we failed to show miR-7 directly regulate AXL and MITF expression (data note shown). [score:5]
Among them miR-7 was found to be the most significantly down-regulated miRNA in VemR A375 melanoma cells. [score:4]
We next tested if the down-regulation of miR-7 was also involved in acquiring resistance to vemurafenib in parental A375 cells. [score:4]
We identified that 17 miRNAs dysregulated in VemR A375 melanoma cells compared to vemurafenib-sensitive parental A375 cells and miR-7 was the most significantly down-regulated miRNA among them. [score:4]
We found that when treated with 100 nM vemurafenib for 72 hrs, parental A375 cells transfected with miR-7 inhibitor showed no significant differences in proliferation as compared with those cells transfected with miR-NC, which suggested that miR-7 exhibited no inhibitory effect on the proliferation of parental A375 melanoma cells when exposed to BRAFi (Figure 1F). [score:4]
miR-7 inhibits VemR A375 melanoma tumor growth in vivo. [score:3]
The results indicated that miR-7 might reverse the resistance to BRAFi in VemR A375 cells by suppressing the activation of both PI3K/AKT and MAPK pathways. [score:3]
We next transfected VemR A375 cells with miR-7 mimics for 48 hrs and found that not only the mRNA expressions of EGFR (p = 0.0402), IGF-1R (p = 0.0021) and CRAF (p = 0.0097) significantly decreased, the protein levels of these genes were also markedly decreased in these cells (Figure 3C and 3D). [score:3]
In the meantime, to verify the underlying mechanism of the inhibitory effect of miR-7 on VemR melanoma tumor growth we also performed the immunohistochemistry (IHC) staining of EGFR, IGF-1R, CRAF, p-ERK and p-AKT and analyzed the total integrated optical density (IOD) of each parameter from both tumor groups. [score:3]
Reestablishment of miR-7 expression could reverse the resistance to BRAFi in both VemR A375 and Mel-CVR melanoma cells. [score:3]
miR-7 suppresses the activation of MAPK and PI3K/AKT pathways in VemR A375 cells. [score:3]
Immunohistochemical staining showing the expressions of EGFR, IGF-1R, CRAF, p-ERK and p-AKT within tumors formed by hypodermic injection of VemR A375 melanoma cells stably transfected with NC or miR-7 mimics. [score:3]
Another important question is whether miR-7 reverses the resistance to BRAFi in melanoma by modulating the expression of EGFR/IGF-1R/CRAF and the activity of their down-stream signaling pathways. [score:3]
de/) to search for miR-7 target genes. [score:3]
miR-7 suppresses the activation of MAPK and PI3K/AKT signaling pathways in VemR A375 cells. [score:3]
These data indicated that elevated miR-7 levels in VemR A375 cells markedly suppressed tumor growth. [score:3]
F. Cell viability of parental A375 melanoma cells stably transfected with miR-NC or miR-7 inhibitor exposed to 100 nM vemurafenib for 72 hrs. [score:3]
Figure 4miR-7 inhibits VemR A375 melanoma tumor growth in vivoVemR A375 melanoma cells (2 × 10 [5]) were transfected with miR-NC or miR-7 mimics and then injected subcutaneously to athymic nude mice (n = 5 for each group). [score:3]
The first question is to identify the target genes of miR-7 that participate in the acquired resistance to BRAFi in melanoma. [score:3]
E. Expression levels of miR-7 in VemR A375 melanoma cells transfected with miR-7 or miR-NC. [score:3]
In addition, we also showed that transfection with miR-7 mimics could significantly suppress the invasion and migration of VemR A375 cells as compared with transfection with miR-7 inhibitor by transwell migration and invasion assays (Supplementary Figure S5A, S5B and S5C). [score:3]
miR-7 inhibits VemR melanoma tumor growth in vivo. [score:3]
The expression levels of EGFR/IGF-1R/CRAF mRNA C. and protein D. in VemR A375 melanoma cells transfected with miR-NC or miR-7 mimics for 48 hrs. [score:3]
Furthermore, tumor growth was inhibited in an in vivo murine VemR A375 melanoma tumor mo del transfected with miR-7 mimics. [score:3]
miR-7 inhibits VemR melanoma tumor growth in vivoTo further investigate whether increased levels of miR-7 could inhibit VemR melanoma growth in vivo, VemR A375 cells were transfected with miR-7 mimics (VemR A375-miR-7) or miR-NC (VemR A375-miR-NC) to produce subcutaneous tumors in athymic nude mice. [score:3]
We first verified a 7.2-fold increase in miR-7 expression after transfected with miR-7 mimics (p < 0.01) compared to miR-NC (Figure 2E). [score:2]
Compared with mock and miR-NC, the expressions of p-AKT and p-ERK in VemR A375 cells transfected with miR-7 mimics were significantly decreased (Figure 2F). [score:2]
In our studies we first used TargetScan and PicTar to screen for candidate genes of miR-7 and compared these candidate genes with those genes that are already known to be involved in the resistance to BRAFi. [score:2]
Nevertheless, these findings only revealed the potential interaction of these two molecules to some extent, further investigation is still needed to explore the underlying molecular mechanism of miR-7 in the regulation of PTEN expression and the subsequent changes of the related signaling pathways. [score:2]
50-70% confluent cells were transfected with human miR-7 mimics or negative control (miR-NC) (GenePharma RNAi Company, Shanghai, China) and 30-40% confluent cells were transfected with EGFR/IGF-1R/CRAF siRNA or siRNA-NC (GenePharma RNAi Company, Shanghai, China) by Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. [score:1]
VemR A375 melanoma cells (2 × 10 [5]) were transfected with miR-NC or miR-7 mimics and then injected subcutaneously to athymic nude mice (n = 5 for each group). [score:1]
Representation (up) and quantification (down) of xenograft tumors formed by subcutaneous injection of VemR A375 melanoma cells stably transfected with miR-NC or miR-7 mimics. [score:1]
We next tested the cell viability of Mel-CVR melanoma cells which were transfected with miR-7 mimics and cultured under 2 uM of vemurafenib for 72hrs. [score:1]
F. Western blot results of p-AKT and p-ERK in VemR A375 melanoma cells transfected with miR-7 or miR-NC. [score:1]
Our study revealed the potential involvement of miR-7 in BRAFi resistant melanoma growth under some circumstances and raised the possibility that this might be exploited therapeutically. [score:1]
Five after transfection of miR-7 mimics or miR-NC vemurafenib resistant A375 cells, the cells were trypsinized, rinsed, and subcutaneously implanted (2 × 10 [5] cells in 100 ul PBS) per flank of nude BALB/c mice (4-week-old males). [score:1]
The tumors in VemR A375-miR-7 group grew more slowly than those in VemR A375-miR-NC group with significantly smaller tumor volumes at day 7 post-implantation (Tumor volumes [miR-7]/Tumor volumes [miR-NC] = 0.5723, p = 0.0116) (Figure 4). [score:1]
To further investigate whether increased levels of miR-7 could inhibit VemR melanoma growth in vivo, VemR A375 cells were transfected with miR-7 mimics (VemR A375-miR-7) or miR-NC (VemR A375-miR-NC) to produce subcutaneous tumors in athymic nude mice. [score:1]
, this may explain why miR-7 can only partially reverse BRAFi resistance. [score:1]
Moreover, miR-7 could also restore the sensitivity of the therapy-resistant cancer cells to different therapies in several cancer types [42- 46]. [score:1]
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[+] score: 239
Intriguingly, miR-7 expression was significantly decreased by these secretagogues (Supplementary Fig. 4), suggesting an unrecognized molecule or pathway that suppressed the expression of miR-7. This inhibitory effect on miR-7 expression, however, is beneficial for insulin secretion because miR-7 overexpression was found to reduce insulin secretion. [score:13]
Cdr1as upregulates miR-7 target genes expressionFurthermore, we examined whether Cdr1as and/or miR-7 are capable of modulating the expression of endogenous Myrip and Pax6. [score:10]
This observation confirmed that the inhibitory role of Cdr1as in miR-7 is associated with the binding of the target, not the degradation of miR-7 6. Since miR-7 interacts with Cdr1as, we examined whether endogenous miR-7 expression level is affected by forskolin, PMA and glucose treatment under the same condition as used in the stimulation of Cdr1as expression. [score:9]
As shown in the working mo del (Fig. 7), we found that Cdr1as or miR-7 expression was upregulated or downregulated respectively by forskolin and PMA, indicating that Cdr1as/miR-7 is involved in the cAMP and PKC signal pathway. [score:9]
This observation confirmed that the inhibitory role of Cdr1as in miR-7 is associated with the binding of the target, not the degradation of miR-7 6. Since miR-7 interacts with Cdr1as, we examined whether endogenous miR-7 expression level is affected by forskolin, PMA and glucose treatment under the same condition as used in the stimulation of Cdr1as expression. [score:9]
Cdr1as upregulates miR-7 target genes expression. [score:8]
Overexpression of exogenous Cdr1as or stimulation of forskolin or PMA can significantly increase the expression level of Cdr1as in islet cells, which in turn inhibits miR-7’s function in insulin biosynthesis and secretion. [score:7]
These findings clearly showed that Pax-6 levels, as a major target of miR-7, is likely to be upregulated by Cdr1as in islet cells. [score:6]
Although many miR-7 target genes were predicted by miRNA software, only a dozen of them have been experimentally demonstrated to be direct targets in insulin pathway of adult islet β cells. [score:6]
MiR-7 (miR-7a is dominantly expressed in islets) expression plasmid, miRVec-miR-7 and its control plasmid (harboring a scrambled sequence, named as “Ctrl1”) were kindly provided by Dr. [score:5]
miR-7 expression level in islet cells, suggesting most if not all of miR-7 will be inhibited by Cdr1as. [score:5]
Myrip and Pax6 are miR-7 targetsTo explore possible molecular interactions of Cdr1as/miR-7 in islet β cells, we analyzed potential miR-7 targets with two wi dely-used bioinformatics tools, PicTar (http://pictar. [score:5]
Similar results were also observed in freshly isolated mouse islets, (Fig. 3b) i. e., ~30% decrease of insulin secretion in miR-7 overexpression group and ~40% increase in Cdr1as overexpression group (Fig. 3b). [score:5]
Since miR-7 is abundantly expressed in islet cells, we assumed that Cdr1as is also expressed in islet cells and other neuroendocrine tissues. [score:5]
In fact, miR-7 expression was reduced in either glucose or forskolin, whereas Cdr1as expression showed positive response to both secretagogues. [score:5]
Decreased insulin content by miR-7 expression or increased insulin protein by Cdr1as expression was confirmed in MIN6 cells and islet cells (Fig. 4c,d). [score:5]
In order to demonstrate that Myrip is a novel direct target of miR-7, we established two dual-luciferase reporter constructs that contain either a wildtype or a mutated 3′-UTR of Myrip. [score:4]
How to cite this article: Xu, H. et al. The circular RNA Cdr1as, via miR-7 and its targets, regulates insulin transcription and secretion in islet cells. [score:4]
Thereby it was ascertained that miR-7 had an inhibitory effect on Myrip and Pax6 through direct binding of their 3′-UTR. [score:4]
In mouse islets cells, however, overexpression of ciRS-7 reached ~90% increase of insulin content compared to the control, while the miR-7 expression resulted in ~20% decrease of insulin content (Fig. 4b). [score:4]
These results demonstrate that miR-7 could regulate the expression of Myrip and Pax6. [score:4]
For the sake of strong effects on miR-7 function, Cdr1as could be an important regulator to prevent miR-7 from interacting with target transcripts in islet cells. [score:4]
We first confirmed that the Cdr1as expression has direct inhibition on miR-7 activity in islet cells by measuring luciferase reporter activities with constructs either containing miR-7 binding site or the entire ciRS-7 sequence (Supplementary Fig. 1). [score:4]
Also, Pax6 (paired box 6) is a transcriptional factor regulating insulin biosynthesis and secretion 33 34, and a target of miR-7 reported previously 29. [score:4]
To explore possible molecular interactions of Cdr1as/miR-7 in islet β cells, we analyzed potential miR-7 targets with two wi dely-used bioinformatics tools, PicTar (http://pictar. [score:3]
Also, the identification of miR-7 target genes related to insulin signaling pathway in islet β cells may reveal the molecular network responsible for the insulin secretion and homeostasis. [score:3]
Expression levels of Cdr1as and miR-7 are normalized to Gapdh mRNA levels. [score:3]
As expected, miR-7 overexpression reduced ~20% insulin secretion in MIN6 cells. [score:3]
Furthermore, we examined whether Cdr1as and/or miR-7 are capable of modulating the expression of endogenous Myrip and Pax6. [score:3]
Using the insulin ELISA assay, we found that ~25% reduction of insulin content in miR-7 overexpressed MIN6 cells compared to a ~70% increase of insulin content in Cdr1as overexpressed MIN6 cells (Fig. 4a). [score:3]
Each of the construct DNAs was co -transfected with miR-7 expression vector or control vector into 293T cells. [score:3]
Since insulin content was increased by the Cdr1as treatment, we examined whether overexpression of Cdr1as and/or miR-7 affects endogenous insulin1 and insulin2 mRNA levels. [score:3]
Concomitantly, the expression pattern of miR-7 was largely similar to that of Cdr1as, but its transcriptional level in islet or pituitary gland was much higher than Cdr1as (Fig. 1a). [score:3]
In addition, the interaction of Cdr1as with miR-7 could be targeted by another miRNA, miR-671, which triggers endonucleolytic cleavage of Cdr1as 25. [score:3]
Altogether, our results indicate that the effect of Cdr1as on insulin content is through insulin biosynthesis, in which potential target genes of miR-7 may actually play an important role. [score:3]
Myrip and Pax6 are miR-7 targets. [score:3]
As we mentioned earlier, transgenic mice overexpressing miR-7 in β cells developed diabetes due to impaired insulin secretion and β cell dedifferentiation. [score:3]
Apparently, the inactivation of miR-7 in mouse islets showed broad effects on insulin pathway due to alterations of hundreds of potential targets. [score:3]
Overexpression of Cdr1as in islet cells was predicted to result in alterations of insulin secretion because of the function of miR-7 as we discussed above. [score:3]
As determined by qRT-PCR, ~30% and ~20% reduction of insulin 1 and insulin 2 gene respectively were observed in the miR-7 overexpressed MIN6 cells (Fig. 5a). [score:3]
Western blots confirmed that miR-7 reduced Myrip and Pax6 protein levels, while Cdr1as dramatically increased their expression in MIN6 cells (Fig. 6c). [score:3]
Therefore, additional miR-7 targets involving insulin granule metabolism as well as insulin homeostasis should be identified. [score:3]
To identify new targets of miR-7 in insulin secretion pathway, we screened hundreds of candidate genes that were predicted by multiple bioinformatic tools and extensively analyzed the candidate’s function in islet cells. [score:3]
In particular, physiological and biological functions of two major target genes (e. g., Myrip and Pax6) of miR-7 have been well studied in islet cells. [score:3]
Similarly, the endogenous Cdr1as expression level was not altered by exogenous transfection of the miRVec-miR-7 (Supplementary Fig. 3b). [score:3]
This finding is in agreement with the inhibitory role of miR-7 on Pax-6 by ours and others 29. [score:3]
Four plasmids, including Cdr1as expression plasmid, pCDNA3-ciRS-7 and its scrambled sequence plasmid (which inserted a Cdr1as sequence only but no invert repeat flanking introns, resulting in no circular Cdr1as production; named as “Ctrl2”), psiCheck-miR-7 and psiCheck-CiRS-7, were kindly given by Dr. [score:3]
While the forced expression of miR-7 in mouse islet cells showed even more decrease of Myrip mRNA (~50%) and Pax6 mRNA (~60%) compared to their controls (Fig. 6b). [score:2]
Whether these responses are derived from associated promoter elements like CREB binding sites or from indirect elements of the Cdr1as/miR-7 network remains to be studied. [score:2]
MiR-7 expression also responded in a similar way to these secretagogues. [score:2]
Forced expression of miR-7 in MIN6 cells was found to result in ~40% decrease of Myrip mRNA and 50% reduction of Pax6 mRNA compared to the control plasmids -treated cells (Fig. 6b). [score:2]
Furthermore, after miR-7 or Cdr1as plasmid DNA was transfected into MIN6 cells and pancreatic islet cells for 48 h, miR-7 or Cdr1as expression was found to be increased ~70 folds or ~180 folds respectively, compared to the control (Supplementary Fig. 2). [score:2]
However, luciferase activities in the mutant Myrip or the mutant Pax6 did not show significant alterations because the mutations within the seed sequence of Myrip or Pax6 abrogated the binding site of miR-7 (Fig. 6a). [score:2]
In particular, Cdr1as, which is derived from an antisense transcript of the CDR1 protein-coding gene at chromosome Xq27.1, contains 71 binding sites or 26 clusters corresponding to miR-7 sites. [score:1]
Cdr1as and miR-7 plasmid DNAs were separately transfected into MIN6 cells and also dissociated mouse islet cells in culture plates (see details in). [score:1]
Furthermore, an interesting observation is that inactivation of miR-7 in obese mice might be sufficient to rescue β cell failure and glycemia 24. [score:1]
Working mo del of Cdr1as/miR-7 -associated network in β cells. [score:1]
Among multiple hits of miR-7, two interesting genes were prioritized because of their role in insulin biosynthesis and exocytosis. [score:1]
We observed that 40% reduction in the wildtype Myrip transfected cells and 50% reduction in Pax6 transfected cells when co -transfected with miR-7, but not co -transfected with control (Fig. 6a). [score:1]
Interestingly, this interaction between Myrip and MyosinVa was activated by cAMP pathway 39, which is inversely correlated with miR-7 by forskolin treatment observed in this study. [score:1]
293T cells were co -transfected with Myrip or Pax6 3′-UTR luciferase reporter plasmid DNA and miRVec-miR-7 or its control plasmid DNAs at 1:10 ratio and were then harvested after 48 h in culture. [score:1]
Importantly, genetic inactivation of miR-7 in β cells was found to result in increased insulin secretion but not affecting proliferation and apoptosis, indicating that miR-7 is dispensable for the maintenance of endocrine β cell mass 24. [score:1]
Islets cells were transfected with plasmid Ctrl2 or pcDNA3-ciRS-7, together with or without miRVec-miR-7 and psiCheck reporter plasmids. [score:1]
After measuring 3′-UTR luciferase reporter activity of the selected candidate gene, we further confirmed that Myrip expression level was decreased by miR-7 but increased by Cdr1as in the islet cells (Fig. 7). [score:1]
In fact, the increased insulin secretion was shown in both acute phase and second phase in the miR-7 deletion mouse 24. [score:1]
However, the endogenous miR-7 expression level, as measured by qRT-PCR, was found to be unaffected by exogenous transfection of the pCDNA3-CiRS-7 (Supplementary Fig. 3a). [score:1]
These results showed that Cdr1as, as a specific repressor of miR-7, is indeed implicated in the insulin pathway. [score:1]
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Other miRNAs from this paper: mmu-mir-7a-1, mmu-mir-7b
Moreover, in vitro and in vivo evidence shows that miR-7, a known microRNA involved in PD via regulating α-Syn, targets Nlrp3 expression and inhibits NLRP3 inflammasome activation in PD mo del mice (Fig.   7). [score:8]
showed that miR-7 mimics significantly down-regulated midbrain NLRP3 expression accompanied by the inhibition of caspase-1 activation and reduction of IL-1β production (Fig.   6j-m), while immunostaining showed miR-7 mimics had no significant effect on TH [+] cell number in A53T [tg/tg] mice at basal state (Fig.   6n & o). [score:8]
j- m Stereotactic injection of miR-7 mimics into the striatum of A53T mice significantly downregulates NLRP3 expression and inhibits production of caspase-1 and IL-1β in vivo. [score:8]
d Transfection of miR-7 into BV2 cells markedly suppresses ATP or MSU triggerd upregulation of NLRP3 and caspase-1 as well as inhibits IL-1β production. [score:8]
These results demonstrate that Nlrp3 is a target gene of miR-7. Fig. 5Nlrp3 is a target gene of microRNA-7. a Luciferase reporter assays confirm that Nlrp3 is a direct target gene of miR-7. Data are presented as the mean ± S. E. M from three independent experiments. [score:7]
miR-7 targets Nlrp3 expression and modulates neuroinflammation in the pathogenesis of PD IL-1β has been regarded as a critical inducer in the pathogenesis of neurodegenerative diseases, including AD and PD [26, 27]. [score:7]
Therefore, we further performed dual luciferase reporter assays, and our result confirms that Nlrp3 is a target gene of miR-7. Moreover, miR-7 mimics effectively inhibited LPS plus ATP/MSU -induced increase in NLRP3 expression and inflammasome activation while anti-miR-7 exacerbated the effect of LPS plus ATP/MSU on NLRP3 inflammasome. [score:6]
c Anti-miR-7 upregulates NLRP3 expression but has no effect on caspase-1 maturation. [score:6]
Taken together, our findings indicate that reduced miR-7 either directly leads to NLRP3 upregulation or indirectly activates NLRP3 inflammasome via α-Syn stimulus during the process of PD. [score:6]
It has been known that miR-7 directly regulates α-Syn expression in DA neurons via post-transcriptional regulation and is associated with the pathophysiology of PD [20]. [score:6]
In agreement with the results of ATP and MSU, miR-7 also can inhibit α-synuclein -induced NLRP3 upregulation and inflammasome activation (Fig.   5f). [score:6]
The results showed that the transfection of miR-7 significantly reduced NLRP3 protein levels (Fig.   5b), whereas anti-miR-7 transfection up-regulated NLRP3 expression (Fig.   5c). [score:6]
f Transfection of miR-7 suppresses WT or A53T α-Syn -induced upregulation of NLRP3 and caspase-1 as well as reduces IL-1β production. [score:6]
We thus presume that miR-7 may directly regulate NLRP3 expression in microglia while regulates α-Syn in neurons. [score:6]
We further unravel that miR-7 targets Nlrp3 expression besides α-Syn and modulates NLRP3 inflammasome -mediated inflammation. [score:5]
We discovered that miR-7 could suppress the expression of Renilla luciferase (R-Luc) through the NLRP3 3′-UTR. [score:5]
It has been demonstrated that microRNA-7 (miR-7), which is expressed mainly in neurons, represses α-Syn protein levels through the 3′-untranslated region (UTR) of α-synuclein mRNA. [score:5]
To further verify our prediction, we inserted the 3′-untranslated region (3′-UTR) sequence of NLRP3 mRNA containing a prominent miR-7 seed sequence into a dual luciferase reporter construct, which allowed us to assess NLRP3 protein translation based on luciferase activities. [score:5]
Notably, we unravel that miR-7 targets Nlrp3 expression besides α-Syn and modulates NLRP3 inflammasome activation. [score:5]
b Transfection of miR-7 into BV2 cells significantly reduces NLRP3 expression but fails to affect procaspase-1 and caspase-1 expressions. [score:5]
Nlrp3 is a target gene of miR-7. miR-7 inhibits NLRP3 inflammasome activation upon the stimulus in vitro. [score:5]
microRNA-7 NLRP3 inflammasome α-Synuclein Neuroinflammation Parkinson’s disease Parkinson’s disease (PD), the second most common neurodegenerative disorder after Alzheimer’s disease, is characterized by the progressive loss of dopaminergic (DA) neurons in substantia nigra compacta (SNc), accumulation of α-synuclein (α-Syn) in Lewy bodies and neurites, and excessive neuroinflammation [1, 2]. [score:5]
Our findings provide a direct link between NLRP3 inflammasome activation and PD pathogenesis, which will give us an insight into the potential of miR-7 and NLRP3 inflammasome in terms of opening up novel therapeutic avenues for neurodegenerative diseases including PD. [score:4]
Therefore, it is also attractive to clarify whether miR-7 could directly modulate microglial NLRP3 inflammasome besides targeting α-Syn. [score:4]
Importantly, miR-7 -induced down-regulation of α-Syn protects DA neurons against oxidative stress [20]. [score:4]
Excitingly, it was predicted that NLRP3 might be a target gene of miR7 by employing the wi dely used program, miRanda [25], suggesting that miR-7 might play an additional role besides regulating α-Syn. [score:4]
Furthermore, α-Syn overexpression (A53T mutation, A53T [tg/tg]) and A53T [tg/tg];Caspase-1 [-/-] double transgenic mice were used to clarify the effects of α-Syn and miR-7 on the activation of NLRP3 inflammasome and neuroinflammation. [score:4]
miR-7 may share the role in regulating NLRP3 expression with other miRNAs in PD pathogenesis. [score:4]
Next, we determined the effect of altered miR-7 or anti-miR-7 on endogenous NLRP3 expression in BV2 cells. [score:3]
e In contrast, miR-7 inhibitor exacerbates inflammasome activation characterized by aggravated NLRP3, caspase-1 expression and IL-1β release upon the stimulus of ATP or MSU. [score:3]
These data further demonstrate that miR-7 protects DA neurons against PD-like degeneration via suppressing NLRP3 inflammasome -mediated neuroinflammation. [score:3]
Most notably, injection of miR-7 mimics dramatically inhibited IBA-1 [+] microglial activation (Fig.   6e) and rescued the loss of TH [+] neuron number in the SNc of MPTP -treated mice (Fig.   6f - g). [score:3]
Most importantly, stereotactic injection of miR-7 mimics markedly inhibited NLRP3 inflammasome -mediated neuroinflammation in mouse brain and protected DA neurons against degeneration. [score:3]
Transfection of miR-7 inhibited microglial NLRP3 inflammasome activation whereas anti-miR-7 aggravated inflammasome activation in vitro. [score:3]
As expected, we found that miR-7 was expressed in both primary microglia and BV2 cells (data not shown). [score:3]
Transfection of miR-7 mimic effectively suppressed caspase-1 activation and subsequent IL-1β production induced by ATP and MSU (Fig.   5d). [score:3]
Thus, it is interesting in test whether microglia expresses miR-7 and altered miR-7 can modulate microglial NLRP3 inflammasome. [score:3]
Specifically, we demonstrated for the first time that Nlrp3 is a target gene of microRNA-7 (miR-7). [score:3]
Interestingly, we find that miR-7 is also expressed in both primary cultured microglia and BV2 cells. [score:3]
Data are presented as the mean ± S. E. M, n = 6. h- i Injection of miR-7 mimics obviously inhibited NLRP3 inflammasome activation in MPTP -treated mice. [score:3]
These findings indicate that α-Syn production is finely controlled by miR-7. In the present study, we show a decreased miR-7 expression in the midbrain of MPTP -induced PD mo del mice, which possibly contributes to increased α-Syn accumulation. [score:3]
It has been known that miR-7 targets α-Syn in DA neurons and is associated with the pathophysiology of PD [20]. [score:3]
When miR-7 seed sequence within the R-Luc-NLRP3-3′-UTR reporter was mutated, the miR-7 -mediated suppression of R-Luc reporter activity was abolished (Fig.   5a). [score:3]
miR-7 mimics suppress NLRP3 inflammasome activation and protects DA neurons against degeneration in PD mo del mice. [score:3]
Meanwhile, miR-7 mimics significantly inhibited NLRP3 inflammasome activation and IL-1β production in MPTP -treated mice (Fig.   6h & i). [score:3]
These results indicate that miR-7 negatively regulates NLRP3 inflammasome activation induced by a variety of stimuli via different pathways. [score:2]
Our study provides a direct link between miR-7 and NLRP3 inflammasome -mediated neuroinflammation in the pathogenesis of PD. [score:2]
For experiments, BV2 cells were transfected with miR-7, anti-miR-7 and corresponding control miRNA for 6 h, followed by stimulation with LPS (100 ng/ml) and then pulsed with 5 mM ATP for 30 min or 250 mg/ml monosodium urate (MSU) for 6 h. Total RNA was prepared using TRIZOL reagent (Invitrogen Life technologies, USA). [score:1]
These findings suggest miR-7 may exert a crucial role in the pathogenesis of PD. [score:1]
e miR-7 dramatically decreases the activation of IBA-1 [+] microglia in WT mice treated with MPTP. [score:1]
Scale bar: 150 μm (f- g) miR-7 mimics remarkedly increase the number of TH [+] neurons in the SNc of WT mice treated with MPTP. [score:1]
miR-7, anti-miR-7 and corresponding control miRNA were complexed respectively with Lipofectamin 2000 (Invitrogen) according to manufacturer’s instructions. [score:1]
Relative miR-7 levels in the serum samples of PD patients (a, n = 12), the midbrain of MPTP -treated mice (b) and A53T [tg/tg] mice (c) as well as α-Syn -treated BV2 cells (d). [score:1]
The effects of miR-7 and anti-miR-7 on NLRP3 inflammasome activation in BV2 cells. [score:1]
Treatment of PD mo del mice with miR-7 mimics. [score:1]
30 nM of miR-7, anti-miR-7 treatment or corresponding NC plus ATP, MSU or α-Syn group To uncover whether miR-7 controls the activity of NLRP3 inflammasomes, we further assessed the effects of miR-7 or anti-miR-7 on ATP- or MSU -induced activation of NLRP3 inflammasomes in BV2 cells. [score:1]
Given that miR-7 could inhibit NLRP3 inflammasome activation in vitro, we thus stereotactically injected miR-7 mimics into wild type mice that treated with subacute MPTP challenge to evaluate the protective effect of miR-7 on DA neurons. [score:1]
Whereas, anti-miR-7 transfection exacerbated MSU or ATP -induced activation of NLRP3 inflammasomes (Fig.   5e). [score:1]
Cells were grown to 70 % confluence in 24-well plates and co -transfected with psiCHECK2-3′UTR plus miR-7 mimics or negative control mimics as described above and previously [36]. [score:1]
Finally, we injected miR-7 mimics into the striatum of A53T [tg/tg] mice to assess its effect on NLRP3 inflammasome in in vivo. [score:1]
Notably, stereotactical injection of miR-7 mimics into mouse striatum attenuated dopaminergic neuron degeneration accompanied by the amelioration of microglial activation in MPTP -induced PD mo del mice. [score:1]
Most importantly, stereotactical injection of miR-7 mimics into mouse striatum attenuated DA neuronal degeneration accompanied by the amelioration of microglial activation in MPTP -induced PD mo del mice. [score:1]
Given that α-synuclein-triggered neuroinflammation plays a crucial role in the pathogenesis of PD, we also detected the effect of miR-7 on NLRP3 inflammasome activation induced by α-synuclein. [score:1]
Moreover, α-synuclein treatment also induced an obvious reduction of miR-7 level in BV2 cells (Fig.   6d). [score:1]
miR-7 mimics had no effect on the numbers of IBA-1 [+] and TH [+] cells in mice without MPTP administration. [score:1]
Briefly, anesthetized mice were positioned in a stereotaxic apparatus, and 2 μl of phosphate-buffered saline containing 0.5 nmol of miR-7 mimics (sense: 5′-UGGAAGACUAGUGAUUUUGUUGU-3′, antisense: 5′-AACAAAAUCACUAGUC UUCCAUU-3′) or a scrambled sequence control miR (sense: 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense: 5′-ACGUGACACGUUCGGAGAATT-3′; GenePharm, Shanghai, China) were injected over 8 min into the striatum (AP: +0 mm, ML: -2.0 mm, DV: -4.0 mm). [score:1]
These findings will give us an insight into the potential of miR-7 and NLRP3 inflammasome in terms of opening up novel therapeutic avenues for PD. [score:1]
Consistently, in the MPTP/p -treated mice and A53T [tg/tg] mice, miR-7 levels were reduced by about 60 % and 55 % respectively, in the midbrain detected by real time PCR (Fig.   6b & c). [score:1]
MiR-7 (sense: 5′-UGGAAGACUAGUGAUUUUGUUGU-3′, antisense: 5′-AACAAAAUCACUAGUCUUCCAUU-3′), control miR (sense: 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense: 5′-ACGUGACACGUUCGGAGAA TT-3′), anti-miR-7 (5′-ACAACAAAA UCACUAGUCUUCCA-3′) and anti-miR control (5′-CAGUACUUUUGUGUAGUACA A-3′) were purchased from GenePharm (Shanghai, China). [score:1]
Specifically, miR-7 is evolutionarily conserved among vertebrates, including mouse and human. [score:1]
n- o immunostaining showed miR-7 mimics had no significant effect on TH [+] cell number in A53T [tg/tg] mice at basal state. [score:1]
Neither miR-7 nor anti-miR-7 transfection altered the protein levels of caspase-1 and IL-1β production (Additional file 4: Figure S4b-S4f). [score:1]
[1 to 20 of 72 sentences]
9
[+] score: 216
Thus, it is the first study showed that miR-7 played an important role in the pathology of ALI, closely correlated with upregulated expression of KLF4, which could ultimately aid the understanding of development of ALI and the development of new therapeutic strategies against clinical inflammatory lung diseases. [score:10]
To elucidate the potential molecular mechanism through which miR-7 deficiency affected the pathologies of lung injury, we searched for putative targets of miR-7 using computer-aided miRNA target prediction program, including TargetScan4, miRanda, and PicTar, and found 12 putative miR-7 target genes, including Snap23, TAB2, KLF4, Smad5, MAPK4, FSTL-1, Tle-4, OGT-1, RAF-1, MAPK8, MAPKAP-1, and PTK2, which also were closely associated with inflammation reaction according to previous literatures (29– 34). [score:9]
Therefore, given the fact that regulation effect of miR-7 on KLF4 expression, these data further highlighted the critical role of miR-7/KLF4 axis in development of ALI, indicating the potential value of this axis in the development of therapeutic strategy against inflammatory lung diseases. [score:8]
Collectively, our data indicated that miR-7 deficiency could affect the pathology of ALI, which was closely due to the upregulation expression of its target KLF4. [score:8]
Of note, KLF4, a target molecule of miR-7, was upregulated in lung tissues, accompanied with reduced expression of NF-κB and phosphorylation of AKT and ERK. [score:8]
Our data showed that the relative expression of miR-7 in various organs, including lung, spleen, brain, and so on, dramatically decreased, indicating miR-7-SP could effectively inhibit the expression of miR-7 in vivo. [score:7]
And KLF4, as one putative target of miR-7, played a critical role in the transduction of these signaling pathways, which were involved in the development of various diseases (55– 59). [score:6]
Importantly, we found that miR-7 deficiency could significantly attenuate the pathologies of ALI, evidenced by accelerated body weight recovery, reduced infiltration of BAL cells and altered level of related cytokines, which was closely correlated to upregulation of KLF4, a target of miR-7, and altered transduction of related signaling pathway, including NF-κB, AKT, and ERK pathway. [score:6]
Recent studies showed that miR-7 was involved in the development of various mammalian diseases, including lung-related diseases (14, 15). [score:6]
Unexpectedly, Real-time PCR assay showed that only one target, KLF4, in all predicted target genes of miR-7 was significantly upregulated by more than 25-fold in the lung tissue of LPS -treated miR-7KD mice compared with LPS -treated WT mice (Figures 4A,B). [score:6]
As shown in Figure 4C, the relative expression of miR-7 increased significantly on day 1, and reached the peak on day 2, then decreased on day 3. Consistently, the relative expression of KLF4 decreased obviously in day 2 and increased significantly on day 3. Next, to verify the role of KLF4 in the effect of miR-7 deficiency on the pathology of ALI, we further detected the protein level of KLF4 in lung tissues from LPS -treated miR-7KD mice and WT mice. [score:5]
To lung cancer, accumulating evidence suggested that miR-7 was an important regulator in the development of lung cancer through controlling the growth and invasion, as well as apoptosis, of lung cancer cells and was emerged as a novel potential therapeutic target in lung cancer. [score:5]
The design and proof of principle of miR-SP technology is to construct a eukaryotic expression vector encoding an expression cassette for miR-7-SP sequence, the miR-7-SP elements included inserting tandemly arrayed miR-7 binding sites into the 3′UTR of a reporter gene encoding destabilized EGFP driven by the CMV promoter. [score:5]
miR-7 deficiency leads to upregulation of KLF4, which could successively alter the transduction of NF-κB, ERK, and AKT signaling pathway and contribute to the ameliorated pathologies of ALI. [score:4]
To address this aim, we generated miR-7 knock down (KD) mice by using miRNAs-Sponge technique and found that miR-7 deficiency could obviously attenuate the pathologies of lung in LPS -induced ALI mice mo del, which related to altered expression of related cytokines and infiltration of innate and adaptive immune cells. [score:4]
MicroRNA-7: a miRNA with expanding roles in development and disease. [score:4]
Moreover, the reduced expression of miR-7 was associated with the sites mutation of its promoter region in lung cancer tissues (17). [score:4]
These data might reflect the important role of miR-7 in the development of lung-related diseases, including ALI. [score:4]
Recent studies showed that KLF4, a critical regulator in LPS -induced inflammatory response (35), was one of targets of miR-7 (36). [score:4]
Therefore, these studies raised a question regarding whether miR-7 also was involved in the development of lung-related inflammatory diseases, including ALI, which remains to be fully elucidated. [score:4]
After 48 h, the relative expression of the potential target genes of miR-7 in the lung tissue were analyzed by Real-time PCR assay. [score:4]
Moreover, our data further showed that, miR-7 not only was enriched in a variety of normal tissues, including brain and lung tissue, which was consistent with previous literature (44– 46), but also was upregulated in lung tissue of LPS -induced ALI mo del. [score:4]
However, the potential role of miR-7 in other lung-related diseases remains largely unknown. [score:3]
Expectedly, we further found that the expression of miR-7 significantly increased in pathogenic lung tissues in LPS -induced ALI mice (Figure S1B in, p < 0.05). [score:3]
Interestingly, Akbas et al. (18) reported that the level of miR-7 was increased and might be a potential biomarker in chronic obstructive pulmonary disease. [score:3]
The relative expression of miR-7 and KLF4 were detected by Real-time PCR at indicated time points. [score:3]
Moreover, Buggele et al. (19) found that influenza A virus infection could induce the expression of miR-7 in human respiratory cells, indicating miR-7 may be involved in the antivirus inflammatory reaction. [score:3]
We, first, observed that the expression of miR-7 increased significantly in lung tissue in murine LPS -induced ALI mo del. [score:3]
Notably, we further reported that altered expression of activated membrane molecular, including CD86 and MHC-II, on F4/80 [+] Mφ cells in LPS -treated miR-7 KD mice. [score:3]
Thus, to elucidate whether miR-7 deficiency resulted in the attenuated pathologies of ALI might be related with these signaling pathway, the expression of phosphorylation of AKT and ERK were analyzed in lung tissue derived from LPS -treated WT mice or miR-7KD mice, respectively. [score:3]
Similarly, Akbas et al. (18) reported that the level of miR-7 was increased and might be a potential biomarker in chronic obstructive pulmonary disease. [score:3]
Importantly, we found that the relative expression of miR-7 was obviously reduced in lung in miR-7KD mice (Figure 1D, p < 0.05). [score:3]
Taken together, as shown in Figure 5D, we presumed that miR-7 deficiency altered that expression of KLF4, which affected the transduction of related signaling pathway, such as NF-κB, AKT, and ERK, and successively influence on the infiltration of various immune cells and the level of inflammatory cytokines, which ultimately ameliorated the pathologies of LPS -induced ALI. [score:3]
Data showed that miR-7 expression had a higher level in lung tissue (Figure S1A in). [score:3]
To confirm these data, we further detected the expression of miR-7 in lung tissues of miR-7KD mice using in situ hybridization and obtained similar results (Figure 1E). [score:3]
Therefore, successive research work on the expression of miR-7 in clinical ALI cases, as well as the potential effect of miR-7 on other animal ALI mo dels that were with distinct traits related to diverse pathological characters of clinical ALI, is much valuable for the validation on the role of miR-7 in pathology of ALI. [score:3]
The sketch map of construction of eukaryotic expression vector encoding miR-7-Sponge (termed as p-miR-7-Sp) (A) and generation of miR-7 KD mice (B). [score:3]
Consistently, the relative expression of miR-7 in other various organs and tissues in miR-7KD mice also decreased significantly (Figure S2 in, p < 0.05). [score:3]
Therefore, our current data might provide valuable clues for the related research work on the potential function of miR-7 in immune cells, which also should be helpful for understanding the role of miR-7 in development of ALI. [score:2]
The expression of miR-7 was performed according to the TaqMan assays and samples were normalized by evaluating U6 expression. [score:2]
Next, we observed the possible effects of miR-7 deficiency on the development of ALI using a murine mo del of LPS -induced ALI. [score:2]
To investigate the potential role of miR-7 in the development of ALI, we detected the relative expression level of miR-7 in murine 12 organs, including heart, lung, brain, kidney, and so on. [score:2]
Generation of miR-7 Knock Down Mice. [score:2]
Together, these data indicated that miR-7 might be involved in the development of ALI. [score:2]
Importantly, we further found that miR-7 deficiency could obviously ameliorate the development of LPS -induced ALI, evidenced by prevention of body weight loss and reduced weight and pathological change of lung tissue. [score:2]
MiR-7 was a distinct miRNA family member and played an important role in the context of health, especially in organ differentiation and development (12, 13). [score:2]
Similarly, our new evidence also showed that miR-7 could regulate the proliferation and metastasis of lung cancer cells in vitro and in vivo (16). [score:2]
MicroRNA-7 compromises p53 protein -dependent apoptosis by controlling the expression of the chromatin remo deling factor SMARCD1. [score:2]
After blocking with normal goat serum (1:100), sections were next incubated or microwave heating and were then incubated with hybridization cocktail containing miR-7 probe (1:1000 dilution; EXIQON; no. [score:1]
We next established miR-7 KD (miR-7KD) FVB/N mice using pEGFP-C2-miR-7 sponge vector with the help of Cyagen Biosciences Inc. [score:1]
Second, the potential role of miR-7 in biological function of immune cells, even was still unknown, also might be contributed to the altered migration of immune cells. [score:1]
Furthermore, we generated miR-7 deficiency mice by using miR-SP technology. [score:1]
Deficiency of miR-7 Ameliorated the Pathologies of ALI. [score:1]
Figure 5 The signaling pathway change of miR-7 deficiency in ALI. [score:1]
, and the TaqMan probes of miR-7 (000386) and U6 (001793) were purchased from Life Technologies, the other reagents were from TAKARA Bio Inc. [score:1]
In the current study, we generated miR-7 deficiency mice using miR-SP technology. [score:1]
miR-7 Deficiency Altered the Transduction of Related Signaling Pathway. [score:1]
miR-7 Deficiency Altered the Composition of Immune Cells in BAL of ALI Mice. [score:1]
Moreover, it is difficult to generate miR-7 deficiency mice using traditional strategies, including TALENs and CRISPR, because of mature miR-7 molecule is transcribed from three different genomic loci on chromosomes 9, 15 and 19, respectively (23). [score:1]
Binding sites for miR-7 seed family were perfectly complementary in the seed region with a bulge at positions 9–12 to prevent RNA interference-type cleavage and degradation of the sponge RNA, the bulge contained six repetitive sequences complementary to miR-7 with mismatches for enhanced stability (Figure 1A). [score:1]
We annealed and ligated, gel purified and cloned oligonucleotides for miR-7 binding sites with 5-nt spacers (one spacer: 5′-CGCG-3′) for 6 bulged sites (one bulged site: 5′-AACAAAATCGAGG- -TCTTCCA-3′), into pEGFP-C2 vector (Invitrogen) digested with HindIII and KpnI enzyme for the construction of pEGFP-C2-miR-7 sponge vector. [score:1]
Then, we used miRNA Sponge (miR-SP) strategy to generate mice with miR-7 deficiency. [score:1]
Figure 1 The generation of miR-7 deficiency mice. [score:1]
Then, to investigate the potential connection between miR-7 and KLF4 in lung injury in ALI, we further detected the relative expression of miR-7 and KLF4 in LPS -treated WT mice at different time points. [score:1]
These results suggested that the evident effect of miR-7 deficiency on the pathology of ALI was closely correlated to the altered transduction of NF-κB and AKT, as well as ERK, signaling pathway (Figure 5D). [score:1]
Combining these data suggested that the change on infiltration and functional molecule of these immune cells are contributed to impaired pathologies of ALI in miR-7 KD mice. [score:1]
Combining these data demonstrated that miR-7 deficiency could obviously ameliorate the pathologies of ALI. [score:1]
Figure 2 miR-7 deficiency ameliorated the pathology of ALI. [score:1]
Figure 3 miR-7 deficiency altered the immune cell composition in BAL of mice from ALI. [score:1]
In summary, our results demonstrate that miR-7 deficiency could significantly affect the composition and function of various immune cells in BAL, which was closely related to ameliorated pathology of lung in ALI mice. [score:1]
Up to now, this is the first study to explore the potential role of miR-7 in the pathologies of ALI. [score:1]
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10
[+] score: 185
Other miRNAs from this paper: mmu-mir-7a-1, mmu-mir-7b
Hypoxia induces the expression of microRNA-7-5p that translationally inhibits the expression of EPAC-1 in endothelial cells, resulting in hyperpermeability and the loss of eNOS activity. [score:9]
EPAC-1 expression levels associated with miR-7-5p expression levels to a high extend (r [2] = 0.464, p < 0.001, Fig.   5c) indicating that the hypoxia -induced expression of miR-7 might underlie the loss of EPAC-1 expression in diabetic retinopathy. [score:9]
Furthermore, we show that the reduction in EPAC-1 expression is associated with the hypoxia -induced expression of microRNA-7, resulting in translational repression. [score:7]
We analyzed the expression of 23 reported miR-7-5p target genes in endothelial cells that express miR-7-5p (Suppl. [score:7]
Here, we show that hypoxia induces the expression of miR-7 by endothelial cells in vitro and in vivo, reducing the expression of EPAC-1. The hypoxia -induced reduction in EPAC-1 levels results in endothelial hyperpermeability and a NO/ROS imbalance and is associated with the development of (diabetic) retinopathy. [score:6]
Moreover, in the Ins2 [Akita] mouse mo del for diabetic retinopathy (DR), EPAC-1 expression is vastly reduced, which coincides with a marked increase in microRNA-7 expression. [score:5]
Data are expressed as mean ± SEM of at least three independent experiments The addition of miR-7 mimics to endothelial cells did not cause a reduction in eNOS expression level (Fig.   4e). [score:5]
In silico analyses were used to identify a link between EPAC-1 expression and microRNA-7-5p in endothelial cells and confirmed by western blot analyses on cells expressing microRNA-7-5p. [score:5]
Hence, we investigated if hypoxia could affect EPAC-1 expression through miR-7. Indeed, hypoxia induced the expression of miR-7 in endothelial cells and transfection of endothelial cells with miR-7 mimics reduced EPAC-1 expression by ~50%. [score:5]
The reduction in EPAC-1 expression is in part due to the hypoxia -induced expression of miR-7. Pharmacological activation of EPAC-1 by forskolin or 8-pCPT antagonizes the hypoxia -induced endothelial dysfunction. [score:5]
Fig.  2Hypoxia induces microRNA-7 -mediated suppression of EPAC-1. a Hypoxia induces the expression of miR-7 by endothelial cells. [score:5]
We recently uncovered that EPAC-1 is targeted by microRNA-7 in human pulmonary smooth muscle cells, and microRNA-7 expression is associated with increased oxidative stress levels [21]. [score:5]
Hypoxia during retinopathy increases the expression of microRNA-7, which in turn reduces the protein availability of EPAC-1. The loss of EPAC-1 in endothelial cells causes endothelial junctional instability and concurrently hyperpermeability, as well as the loss of eNOS expression and eNOS activity, resulting in oxidative stress. [score:5]
These data indicate that EPAC-1 is a specific target of miR-7. Corroboratively, in endothelial cells transfected with miR-7 mimics EPAC-1 protein expression was decreased 2.1-fold (p < 0.05, Fig.   2d). [score:5]
Data are expressed as mean ± SEM of at least three independent experiments Endothelial cells exposed to 2% oxygen for 24 h increased miR-7 expression by 9.1-fold (p < 0.01, Fig.   2a). [score:5]
Data are expressed as mean ± SEM of at least three independent experiments The addition of miR-7 mimics to endothelial cells did not cause a reduction in eNOS expression level (Fig.   4e). [score:5]
Remarkably, treating miR-7 -expressing cells with 8-pCPT reduced endothelial hyperpermeability (1.7-fold, p < 0.05), indicating that activation of remnant EPAC-1 is sufficient to inhibit miR-7 -induced endothelial hyperpermeability. [score:5]
To confirm that EPAC-1 and EPAC-2 are genuine targets of miR-7, we produced reporter constructs wherein the expression of luciferase is under the control of the 3′UTR of EPAC-1 or EPAC-2. Co-transfection of COS7 cells with miR-7 mimics and the EPAC-1 reporter construct decreased luciferase activity (2.6-fold) compared to scrambled controls (p < 0.05), whereas the luciferase activity of the EPAC-2 reporter was unaffected (Fig.   2c). [score:4]
As our data implies that miR-7-5p underlies hyperpermeability in oxygen -induced retinopathy and eNOS uncoupling in diabetic retinopathy, inhibition of miR-7-5p, or EPAC-1 activation seem a promising approach to restore retinopathy. [score:3]
e Association between the EPAC-1 and miRNA-7-5p expression levels in non-diabetic and diabetic Ins2Akita mice. [score:3]
Conversely, c hypoxia increases retinal microRNA-7-5p expression at 6 and 24 h. d analysis and e western blot analysis for EPAC-1 in vitro. [score:3]
e miR-7 mimics decrease eNOS activity under normoxic conditions without affecting eNOS expression level. [score:3]
e miR-7 mimics increase endothelial monolayer permeability under normoxic conditions without affecting f VE-cadherin expression. [score:3]
f Levels of phosph-eNOS (Ser1177) and eNOS in non-diabetic and diabetic Ins2Akita mice Besides EPAC-1 [21], miR-7 targets a number of additional gene transcripts (Suppl. [score:3]
Interestingly, hypoxia -mediated endothelial hyperpermeability was associated with a decreased in VE-cadherin expression, whereas hyperpermeability induced by miR-7-5p mimics was not. [score:3]
In mouse mo dels of retinopathy, i. e., oxygen -induced retinopathy and the spontaneous diabetic heterozygous Ins2 [Akita] mice, EPAC-1 levels are decreased which is associated with an increase in microRNA-7-5p expression and reduced eNOS activity. [score:3]
In vitro, endothelial cells were either incubated at 2% oxygen or transfected with microRNA-7-5p, and the effects of these treatments on EPAC-1 expression, endothelial hyperpermeability and NO production were assessed. [score:3]
In the Ins2Akita mouse mo del, levels of EPAC-1 expression as well as microRNA-7-5p were assessed by qPCR. [score:3]
For microRNA expression analyses, 20 ng total RNA was reversely transcribed using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) using specific stemloop templates for miRNA-7-5p (5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAACAAA-3′) or RNU6 (5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAAATATGG-3′) and amplified using sense 5′-TGCGGTTGGAAGACTAGTGAT-3′, antisense 5′-CCAGTGCAGGGTCCGAGGTCCG-3′ for miR-7-5p, and sense 5′-TGCGGCTGCGCAAGGATGA-3′, antisense 5′-CCAGTGCAGGGTCCGAGGTCCG-3′ for RNU6. [score:3]
Corroboratively, inhibition of EPAC-1 activity with the small molecule ESI-09 or miR-7-5p mimics increases the endothelial permeability. [score:3]
f Levels of phosph-eNOS (Ser1177) and eNOS in non-diabetic and diabetic Ins2Akita mice Besides EPAC-1 [21], miR-7 targets a number of additional gene transcripts (Suppl. [score:3]
Indeed, supplementing endothelial cells with miR-7 mimics induced hyperpermeability (Fig.   3e) in the absence of hypoxia and without alterations to VE-cadherin expression (Fig.   3f). [score:3]
In retinopathy, EPAC-1 expression is decreased in a microRNA-7 -mediated manner, contributing to endothelial dysfunction. [score:3]
We had previously found an association between EPAC-1 and miR-7 expression levels in airway smooth muscle cells [21]. [score:3]
Hypoxia induces microRNA-7 -mediated suppression of EPAC-1. cAMP signaling counteracts hypoxia -induced endothelial hyperpermeability. [score:3]
MicroRNA-7 contains a seed sequence that has complementarity to the 3′Untranslated Region (UTR) of EPAC-1 and EPAC-2. EPAC-1 has five putative miR-7 binding sites, whereas EPAC-2 only has one putative miR-7 binding site (Fig.   2b). [score:3]
MiR-7 mimics decrease EPAC-1 protein expression in cultured endothelial cells. [score:2]
d MicroRNA-7-5p expression in retinal lysates of spontaneous diabetic Ins2Akita [+/−] and control mice. [score:2]
MiR-7-5p was detected by in situ hybridization in the retinae from diabetic Ins2Akita mice (Fig.   5c), where its expression of miR-7 was increased (3.2-fold, p < 0.01, Fig.   5d) compared to non-diabetic controls. [score:2]
c Luciferase reporter assays for miR-7:3′UTR binding for EPAC-1 and EPAC-2. EPAC-1 is a genuine target of miR-7. d Immunoblotting of EPAC-1 in endothelial cells transfected with miR-7 mimics or scrambled sequences. [score:2]
Therefore, we hypothesized that microRNA-7 might induce EPAC-1 deregulation during retinal hypoxia or in diabetic conditions. [score:2]
After 6 h and 24 h of relative hypoxia, retinal EPAC-1 gene transcript levels were reduced (2.0- and 1.9-fold, respectively, p < 0.05, Fig.   1b Conversely, miR-7 expression was increased in the retina of OIR-mice (2.7- and 3.2-fold, p < 0.01) compared to normoxic control mice (Fig.   1c). [score:2]
EPAC-1 Hypoxia MicroRNA-7 Endothelial cell Retinopathy The vascular endothelium exhibits multiple structural and functional abnormalities in response to hypoxia that may contribute to the pathogenesis of several vascular diseases, including (diabetic) retinopathy [1]. [score:2]
Endothelial cells were transfected using microRNA-7 mimics and scrambled sequences using EndoFectin (GeneCopoeia, Rockville, MD). [score:1]
c In situ hybridization using scrambled probes or miR-7-5p-specific probes on retinal digests from 6-month-old spontaneous diabetic heterozygous Ins2Akita [+/−] and control non-diabetic littermates. [score:1]
b In silico analysis of the 3′UTR of EPAC-1 and EPAC-2. EPAC-1 has 5 putative miR-7 binding sites, whereas EPAC-2 has one putative miR-7 binding site. [score:1]
In control C57BL/6 mice, miR-7-5p levels remained below the detection limit for in situ hybridization (Fig.   5c). [score:1]
Activation of the remnant EPAC-1 by forskolin or the cAMP analogue 8-pCPT antagonizes the hypoxia or miR-7 -induced endothelial hyperpermeability. [score:1]
Co-transfection of miR-7 mimics and an 3′UTR-free psiCHECK-2 plasmid were used as controls. [score:1]
Moreover, endothelial cells that were transfected with miR-7-5p mimics showed decreased eNOS activity and eNOS activity is decreased in the retinae of diabetic Ins2Akita mice. [score:1]
EPAC-1 and microRNA-7 alterations in diabetic retinopathy. [score:1]
Although we cannot fully exclude that retinal microvasculature endothelial cells would behave different with respect to miR-7-5p or EPAC-1 stimulation, in preliminary experiments we have found no difference between the umbilical vein endothelial cells and dermal microvascular endothelial cells (data not shown). [score:1]
Construction of 3′UTR reporter constructs and microRNA-7-5p transfection in COS7 and Endothelial cells. [score:1]
However, the addition of miR-7 mimics to endothelial cells did imitate the hypoxia -induced loss of eNOS activity as indicated by a reduction in eNOS phosphorylation at serine 1177 (p-eNOS/eNOS ratio; 1.7-fold decrease, p < 0.01; Fig.   4e). [score:1]
Data are expressed as mean ± SEM of at least three independent experiments We next investigated if the addition of miR-7 mimics to endothelial cells would imitate the hypoxia -induced endothelial hyperpermeability. [score:1]
Fig.  6 Hypoxia -mediated repression of EPAC-1 by microRNA-7 in retinopathy. [score:1]
g The miR-7 -induced endothelial hyperpermeability is antagonized by the EPAC-1 activator ESI-09. [score:1]
Data are expressed as mean ± SEM of at least three independent experiments We next investigated if the addition of miR-7 mimics to endothelial cells would imitate the hypoxia -induced endothelial hyperpermeability. [score:1]
[1 to 20 of 58 sentences]
11
[+] score: 166
Our data showed that OGD could downregulate miR-7 (Fig.   2a) and upregulate mRNA level of Herpud2 (Fig.   2b); it motivated ER stress via upregulating ER stress proteins including GRP78, CHOP, and Caspase-12 (Fig.   4a– d). [score:10]
Moreover, we also found that both transfection with miR-7 inhibitor (40 pM) and mimic (40 pM) altered expression of Herpud2 mRNA (Fig.   3c); this was further evidenced when miR-7 regulated the expression of Herpud2 mRNA. [score:8]
We noted that transfection with miR-7 inhibitor or mimic could result in decrease or increase of miR-7. Transfection with 20- or 40-pM inhibitor could respectively induce downregulation of miR-7 by 33 and 45 % (Fig.   3a). [score:8]
Pretreatment with nicorandil could remarkably upregulate miR-7, depress the ER-related protein expressions including glucose-regulated protein 78 (GRP78), C/EBP-homologous protein (CHOP), and Caspase-12, and thereby attenuate inflammatory responses and astrocytic damages. [score:7]
Notably, we found that OGD significantly downregulated miR-7 and upregulated Herpud2. [score:7]
demonstrated that expression of miR-7 in astrocytes was obviously downregulated by about 40 % since OGD for 5 h and reoxygenation for 24 h (Fig.   2a). [score:6]
Kefas B, Godlewski J, Comeau L, Li Y, Abounader R, Hawkinson M, Lee J, Fine H, Chiocca EA, Lawler S, Purow B. microRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway and is down-regulated in glioblastoma. [score:6]
OGD group MiR-7 targets the 3′UTR of Herpud2MiR-7 is proved to be involved in cancer, diabetes mellitus, and Parkinson’s disease via targeting inflammation-related proteins. [score:6]
To further study whether nicorandil affected expression of miR-7 and the target, we pretreated astrocytes with nicorandil and found that pretreatment with nicorandil (10 μM) could reverse OGD -induced decrease of miR-7 (Fig.   2d) and increase of Herpud2 mRNA (Fig.   2e). [score:5]
OGD group MiR-7 alters expression of Herpud2 and concerned with the protection of nicorandil against OGD -induced injuryTo further verify the effects of miR-7, we established miR-7 mimic and inhibitor and transfected them into primary cultured astrocytes. [score:5]
Our study proved miR-7 targeted the important ER stress relevant protein-HERP2, and nicorandil protected against OGD -induced injury depending on miR-7. To further verify whether nicorandil affected OGD -induced ER stress, we detected the expressions of three key important ER stress-related proteins, including molecular chaperon (GRP78/Bip), transcription factor (CHOP), and apoptosis-related protein (Caspase-12). [score:5]
Pretreatment with nicorandil could upregulate miR-7 and alleviate OGD -induced ER stress and inflammatory injuries in astrocytes. [score:4]
OGD group MiR-7 is proved to be involved in cancer, diabetes mellitus, and Parkinson’s disease via targeting inflammation-related proteins. [score:4]
To validate whether miR-7 really targeted the 3′UTR of Herpud2, we constructed Herpud2-3′UTR reporter and used dual luciferase target validation assays to confirm the hypothesis. [score:4]
These results implied that nicorandil protected against OGD -induced injury might be related to miR-7. Fig. 2Nicorandil restrains OGD -induced upregulation of Herpud2 mRNA via miR-7. Time-course of OGD induced the changes of miR-7 (a) and Herpud2 mRNA (b) in astrocytes. [score:4]
MiR-7 is a proven small molecule that implicates in inflammation in various diseases [35], such as neurodegenerative disease and glioma [11, 36– 38]. [score:4]
As we proved that pretreatment with nicorandil protected against OGD -induced injury and changed expression of miR-7, to further study whether the protection of nicorandil depended on miR-7, we transfected primary astrocytes with miR-7 inhibitor before pretreatment with nicorandil and detected cell viability by MTT assay. [score:4]
These results suggest that miR-7 may be a potential target for the anti-inflammation effect of nicorandil. [score:3]
Therefore, these results demonstrated that miR-7 targeted the 3′UTR of Herpud2. [score:3]
OGD group To further verify the effects of miR-7, we established miR-7 mimic and inhibitor and transfected them into primary cultured astrocytes. [score:3]
The expression of miR-7 was normalized using U6 as the internal control. [score:3]
org) that miR-7 could potentially target the 3′UTR domain of Herpud2 (HERP2), which was one of the HERP family members, acting as an important ER stress-related molecule. [score:3]
Interestingly, as shown in Fig.   3d, pretreatment with nicorandil (10 μM) could reverse OGD -induced decrease of cell viability; however, transfection with miR-7 inhibitor abolished this effect. [score:3]
Further studies showed for the first time that miR-7 targeted the 3′UTR of Herpud2, which might be crucial for OGD -induced ER stress and inflammation in astrocytes. [score:3]
Potentially, miR-7 -targeted ER stress acts as a key molecular brake on neuroinflammation. [score:3]
Dose response of miR-7 level in primary cultured astrocytes after transfection with miR-7 mimic (a) and/or inhibitor (b). [score:3]
Kong D, Piao YS, Yamashita S, Oshima H, Oguma K, Fushida S, Fujimura T, Minamoto T, Seno H, Yamada Y. Inflammation -induced repression of tumor suppressor miR-7 in gastric tumor cells. [score:3]
c Herpud2 mRNA level in primary cultured astrocytes transfected with miR-7 mimic and inhibitor. [score:3]
Moreover, this targeting effect was specific because there was no significant change of the relative luciferase activity on condition of co-transfection of Herpud2 Mut 3′UTR with miR-7 mimics (Fig.   2c). [score:3]
Expression of miR-7 (d) and Herpud2 mRNA (e) pretreated by nicorandil. [score:3]
d Cell viability of astrocytes after pretreatment with nicorandil and (or) miR-7 inhibitor. [score:3]
Our further study revealed for the first time that OGD decreased inflammation -associated microRNA 7 (miR-7) but increased the messenger RNA (mRNA) levels of Herpud2, one of the miR-7’s targets. [score:3]
Our further study proved that miR-7 targeted Herpud2 3′UTR, which encoded endoplasmic reticulum (ER) stress protein-HERP2. [score:3]
In the present study, we found that miR-7 targeted the 3′UTR of Herpud2 (Fig.   2c). [score:3]
These results suggested that K-ATP channel’s opener alleviated OGD -mediated ER stress and inflammation via regulating miR-7 and thereby protected astrocytes against OGD -induced damage. [score:2]
MiR-7 targets the 3′UTR of Herpud2. [score:2]
MiR-7 alters expression of Herpud2 and concerned with the protection of nicorandil against OGD -induced injury. [score:2]
c The target of miR-7 to Herpud2 3′UTR via Dual Luciferase Reporter Assays. [score:2]
Nicorandil miR-7 Neuroinflammation Oxygen-glucose deprivation Astrocytes are specialized and most numerous glial cell types in the brain, they play crucial roles in central nervous system (CNS) homeostasis [1]. [score:1]
While we used transfection with 40-pM miR-7 mimic, the result demonstrated that it could lead to an increase of about 62-fold in miR-7 level (Fig.   3b). [score:1]
A construct containing the 3′UTR of Herpud2 mRNA or the sequence with the mutant seed region was co -transfected along with miR-7 mimics or mimics NC into HEK293T cells. [score:1]
Thus, our study suggested that miR-7 might be an important upstream sign that modulate ER -associated degradation. [score:1]
indicated that nicorandil protected against OGD -induced injury depending on miR-7. Fig. 3MiR-7 is indispensable for the protection of nicorandil against OGD -induced injury. [score:1]
In the present study, pretreatment with nicorandil (a K-ATP channels’ opener) could rescue the reduction of miR-7 induced by OGD (Fig.   2d). [score:1]
Our study revealed that miR-7 was also involved in OGD -induced ER stress and inflammatory responses in astrocytes. [score:1]
When cells were grown to 50 % confluence in 96-well plates, they were co -transfected with 0.2 μg plasmid DNA, 0.15 μg sensor reporter gene, and 0.45 μg miR-7 mimics or miRNA negative control (NC) (Gemma Pharmaceutical Technology Co. [score:1]
Thus, we determined the time-course of Herpud2 mRNA level and miR-7 via real-time PCR. [score:1]
, Ltd, Shanghai) used for real-time PCR are listed in Table  1. The 3′UTRs of Herpud2 mRNA harboring the predicted miR-7a binding sequences were PCR amplified from mouse genomic DNA and cloned into Bam HI and Xho I of the pLUC-Report luciferase vector (Shenzhen Kangbio Biological Technology Co. [score:1]
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12
[+] score: 156
For miR-7 regulated expression of TINCR, TINCR full length of transcript (wild-type, miR-7 target region deletion, miR-7 target region mutation) was fused to luciferase, and co -transfected MCF-7 or MDA-MB-468 cells with miR-7a/b. [score:9]
a The relative expression of miR-7 in response to TINCR knockdown or miR-7 specific inhibitor was determined by real-time PCR in both MCF-7 and MDA-MB-468 cells. [score:6]
Consistent with previous results, TINCR-knockdown significantly suppressed breast cancer cell proliferation, while simultaneous inhibition of miR-7 in this setting almost abolished this effect (Fig. 6b, c). [score:6]
Consistent with these observations, our data suggested that inhibitory effect on KLF4 expression of miR-7 was significantly mitigated by TINCR in breast cancer via competitive mechanism. [score:5]
Several studies implied that KLF4 was the potential target of miR-7. Along this line, here we further investigated the potential that TINCR modulated KLF4 expression via competing with miR-7. The putative target site in 3’UTR region of KLF4 aligned with miR-7 seed region based on the micrRNA. [score:5]
Our data further experimentally confirmed the direct interaction between miR-7 and TINCR, which underlaid its competitive regulation of miR-7 target genes. [score:5]
miR-7 inhibition abrogated TINCR-silencing elicited tumor suppressive effect. [score:5]
Most notably, several studies indicated that miR-7 functioned as tumor suppressor gene via direct regulation of KLF4. [score:5]
This inhibitory effect was readily reversed by co-introduction of either TINCR or miR-7 specific inhibitor (Fig. 5g). [score:5]
Our data suggested a crucial role of TINCR-miR-7-KLF4 axis in human breast cancer and up-regulation of ceRNA TINCR by SP1 contributes to tumorigenesis in breast cancer. [score:4]
Moreover, miR-7 was shown to arrest cell cycle in G1 phase by directly targeting CCNE1 in human hepatocellular carcinoma cells [27]. [score:4]
We employed LncRNABase online algorithm to predict the candidate miR with the potential to directly compete with TINCR, and identified miR-7 as the top one in the putative targets list. [score:4]
For instance, miR-7 has been demonstrated to function as a tumor-suppressor gene in pancreatic carcinoma via regulation of ILF2 [40]. [score:4]
MiR-7 also suppressed cell proliferation and induced apoptosis of breast cancer cells predominately by targeting REGγ [25]. [score:4]
The endogenous expression of miR-7 in response to TINCR knockdown was analyzed in both MCF-7 and MDA-MB-468 cells. [score:4]
On the other hand, inhibition of miR-7 was shown to promote angiogenesis in human umbilical vein endothelial cells by up -regulating VEGF via KLF4 [22]. [score:4]
Either deletion or mutation introduced into the suspected regions of TINCR abolished miR-7 -inhibited luciferase activities (Fig. 5b, c, d, e). [score:4]
Chang et al. reported that miR-7 inhibited the tumorigenesis and stemness of prostate cancer via repressing KLF4/PI3K/Akt/p21 pathway [41]. [score:3]
Exogenous scramble, miR-7 a/b, TINCR or anti-miR-7 were transfected into MCF-7 (left) and MDA-MB-468 cells in combination as indicated, the relative expression of KLF4 was determined by immunoblotting. [score:3]
Okuda et al. reported that miR-7 capable of suppressing brain metastasis of breast cancer stem-like cells by modulating KLF4 [23]. [score:3]
Exogenous introduction of miR-7a/b remarkably inhibited endogenous KLF4 in our immunoblotting results in both MCF-7 and MDA-MB-468 cells. [score:3]
Consistently, the transcripts of KLF4 were decreased in response to miR-7a/b, and subsequently restored by ectopic TINCR or miR-7 inhibitor treatment (Fig. 5h). [score:3]
f The putative target sites of miR-7 in KLF4 3’UTR by microRNA online tool. [score:3]
The expression status of SP1-TINCR-miR-7-KLF4 was further characterized in xenograft tumor at both transcriptional and translational level (Fig. 5i), which definitely consolidated our in vitro observations. [score:3]
Similarly, the reduction in colony formation capacity elicited by TINCR silencing was readily restored while miR-7 was specifically inhibited (Fig. 6d). [score:3]
In line with this notion, in this study we performed bioinformatic analysis to identify miR-7 as one of TINCR targets. [score:3]
g miR-7 negatively modulated KLF4 expression, which was antagonized by TINCR. [score:3]
High level of TINCR in turn competed with miR-7, and stabilized and promoted KLF4 expression, which consequently contributed to the oncogenic activity of TINCR. [score:3]
However, the opposite conclusion emerged as well in especially lung cancer [39], suggested both oncogene and tumor suppressor roles of miR-7 probably in an organ context -dependent manner. [score:3]
As shown in Fig.   6a, TINCR-silencing induced more than 3-fold increase of miR-7, which was completely abrogated by co-treatment with miR-7 inhibitor. [score:3]
Mechanistically, TINCR modulated KLF4 expression via competing with miR-7, which consequently contributed to its oncogenic potential. [score:3]
Most importantly, our data suggested that either specific inhibition of TINCR or complement with miR-7 likely held great promise for breast cancer therapeutics. [score:3]
The candidate target genes of miR-7 involving in tumor biology have been extensively identified and systematically reviewed by Gu et al. [20]. [score:3]
TINCR functioned as endogenous competing lncRNA of miR-7 and involved in KLF4 regulation. [score:2]
Fig. 5TINCR functioned as endogenous competing lncRNA against miR-7 to regulate KLF4. [score:2]
Our previous data suggested that TINCR functioned as competing endogenous RNA (ceRNA) to compete with miR-7 in regulation of KLF4. [score:2]
MiR-7 is conventionally considered as a tumor suppressor miRNA in variety of human malignancies including breast cancer [24], brain cancer [21], liver cancer [38], colon cancer [26]. [score:2]
MiR-7 inhibition severely compromised TINCR silencing-elicited tumor repressive effects. [score:2]
We further experimentally validated the putative binding sites of miR-7 on TINCR transcript. [score:1]
a The prediction of miR-7 seeding region in TINCR transcript using Starbase online tool. [score:1]
Either wild-type or putative binding site deleted TINCR was fused to luciferase plasmid, which was co -transfected with miR-7a/b into MDA-MB-468 and MCF-7 cells. [score:1]
Our results clearly demonstrated the critical role of TINCR in tumorigenesis and metastasis-related malignant behavior in breast cancer, and predominant role of miR-7 in mediating this effect. [score:1]
The exogenous introduction of miR-7 caused about 75% reduction in MDA-MB-468 and 60% reduction in MCF-7 of TINCR-fused luciferase activity (Fig. 5b, c). [score:1]
TINCR miR-7 KLF4 Breast cancer Long non-coding RNA Breast cancer is one of the most common malignancies in women [1]. [score:1]
In line with previous observations, the direct binding of miR-7 with TINCR was experimentally validated in our luciferase reporter assay. [score:1]
The alignment between TINCR and miR-7 was shown in Fig.   5a. [score:1]
Our data uncovered a crucial role of TINCR-miR-7-KLF4 axis in human breast cancer. [score:1]
Our data unambiguously demonstrated that miR-7 predominately involved in oncogenic activity of TINCR in breast cancer. [score:1]
Either wild-type or putative binding site mutant TINCR was fused to luciferase plasmid, which was co -transfected with miR-7a/b into MDA-MB-468 and MCF-7 cells. [score:1]
b, c Luciferase reporter assay was performed to validate the regulatory effect of miR-7 on TINCR. [score:1]
d, e Luciferase reporter assay was performed to validate the regulatory effect of miR-7 on TINCR. [score:1]
Range of investigations have uncovered the important roles of miR-7 in human malignancies with increased list of target genes have been identified [20– 27]. [score:1]
Taken together, our data suggested that TINCR might function as molecular sponge of miR-7, which eventually contributed to its oncogenic activity. [score:1]
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[+] score: 126
By contrast, in the up-regulated genes of 23DSI, the predicted target genes of miR-1-miR-71-miR-7-miR-7-5p appeared to regulate the ribonucleoprotein complex assembly, cellular protein complex assembly, microtubule -based process, response to oxidative stress, multicellular organismal aging, respiratory electron transport chain, pyrimidine ribonucleoside triphosphate biosynthetic process, positive regulation of epithelial cell differentiation, positive regulation of cell proliferation, apoptosis, energy coupled proton transport, electron transport chain, ATP synthesis-coupled proton transport, anatomical structure formation involved in morphogenesis, ribonucleoprotein complex biogenesis, mitotic cell cycle, larval development, microtubule polymerisation or depolymerisation, female gamete generation, regulation of transcription from RNA polymerase II promoter, and imaginal disc development, among others (Table  2 and Additional file 6: Table S4). [score:12]
In 23 DSI, the high level of bantam and low levels of miR-1, miR-71, miR-7, and miR-7-5p possibly regulated and organised a specific gene expression profile for sexual maturation and egg production by inhibiting and strengthening specific gene expression and metabolic processes. [score:8]
In unpaired females (23SSI), bantam was notably not up-regulated, whereas miR-1, miR-71, miR-7, and miR-7-5p were significantly up-regulated. [score:7]
Furthermore, among all samples, bantam was distinctly up-regulated in 23 DSI, and miR-1, miR-71, miR-7-5p, and miR-7 were distinctly up-regulated in 23SSI. [score:7]
To analyse the effect of the differential expression of miRNAs on female development after pairing, we sequenced the libraries of 23DSI and 23SSI, predicted the target genes of miRNA-1-miRNA-71-miRNA-7-miR-7-5p (Additional file 3: Table S1) and bantam (Additional file 4: Table S2), and analysed the differential expression of these genes in 23DSI compared with 23SSI. [score:7]
Although miRNAs do not regulate all genes in organisms, evidence provided by miRNA analyses in the present study indicated that pairing likely limited the expression of non-essential genes through increasing the expression of bantam and specific genes by maintaining miR-1, miR-71, miR-7, and miR-7-5p at relatively low levels. [score:6]
Click here for file Predicted target genes of miR-1-miR-71-miR-7-miR-7-5p in up-regulated genes in 23DSI. [score:6]
Predicted target genes of miR-1-miR-71-miR-7-miR-7-5p in up-regulated genes in 23DSI. [score:6]
By contrast, in paired females (23DSI), the above mentioned miRNAs were not up-regulated, suggesting that the functions of the target genes of miR-1-miR-71-miR-7-miR-7-5p were required in paired females. [score:6]
We found that the target genes of miR-1-miR-71-miR-7-miR-7-5p, such as ribosomal protein genes (CAX72037.1, CAX71939.1, CAX78482.1, CAX77178.1, AAP06483.1, CAX77387.1, CAX72859.1, CAX70956.1, CAX71543.1, CAX83047.1, CAX70121.1) (Additional file 6: Table S4), thioredoxin peroxidase (CAX75860.1), tubulin (XP_002580033.1, CAX75788.1, CAX75500.1, CAX71989.1, CAX76110.1), ATP synthase- H + transporting (CAX75390.1, CAX76063.1), and cytochrome c oxidase (CAX74747.1, CAX76589.1), among others, were significantly up-regulated. [score:6]
Out of the 50 genes, 33 were the predicted target genes of bantam (Figure  3B), whereas only 2 were predicted target genes of miR-1-miR-71-miR-7-miR-7-5p. [score:5]
revealed that in unpaired females, the highly-expressed miRNA-1, miRNA-71, miRNA-7, and miR-7-5p only inhibited the limited pathways, such as proteasome and ribosome assembly. [score:5]
For example, the higher expression of bantam was observed only in 23DSI, whereas higher expression of miR-1, miR-71, miR-7-5p, and miR-7 manifested only in 23SSI (Figure  1B). [score:5]
The predicted target genes of bantam hardly participated in the proteasome, porphyrin metabolism, ribosome, whereas more predicted target genes of miR-1-miR-71-miR-7-miR-7-5p were involved in these process. [score:5]
For instance, in ribosome assembly, 15 of 49 detected genes in this metabolic process were predicted as the target genes of miR-1-miR-71-miR-7-miR-7-5p, whereas only 1 of 49 genes was the predicted target gene of bantam (Figure  3A). [score:5]
Differential expression of the predicted target genes of bantam and miRNA-1-miRNA-71-miRNA-7-5p- miR-7 between samples from 23 DSI and 23SSI. [score:5]
Moreover, few of the predicted target genes of miR-1-miR-71-miR-7-miR-7-5p participated in the peroxisome, RNA degradation, mRNA surveillance pathway, axon guidance, basal transcription factors, apoptosis, glycerophospholipid metabolism, insulin signalling pathway, lysosome, regulation of actin cytoskeleton, and endocytosis. [score:4]
Click here for file Predicted target genes of miR-1-miR-71-miR-7-miR-7-5p in Schistosoma japonicum. [score:3]
Predicted target genes of miR-1-miR-71-miR-7-miR-7-5p in Schistosoma japonicum. [score:3]
However, none of the predicted target genes of miR-1-miR-71-miR-7-miR-7-5p are involved the citrate cycle, gastric acid secretion, glycolysis/gluconeogenesis, protein digestion and absorption, aminoacyl-tRNA biosynthesis, fatty acid biosynthesis, and the pentose phosphate pathway. [score:3]
The transcriptomes of 23DSI and 23SSI revealed that the predicted target genes of miRNA-1, miRNA-71, miRNA-7, and miR-7-5p were associated with the ribonucleoprotein complex assembly and microtubule -based process. [score:3]
Only several high-abundance miRNAs differentially expressed between 23DSI and 23 SSI, such as bantam, miR-1, miR-71, miR-7, and miR-7-5p. [score:3]
In particular, various ribosomal protein genes were regulated by miR-1-miR-71-miR-7-miR-7-5p. [score:2]
Furthermore, the low abundance of miR-1, miR-71, miR-7, and miR-7-5p in 23DSI compared with 23SSI was likely capable of promoting specific gene expression. [score:2]
These results suggested that miR-1, miR-71, miR-7, and miR-7-5p played an essential role in regulating ribosomal assembly. [score:2]
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[+] score: 110
Because Klf4 expression was promoted by CHIR, we asked whether CHIR enhanced Klf4 expression by repressing the level of miR-7a that targeted Klf4. [score:7]
Thus, CHIR inhibits miR-7a expression, likely by inhibiting GSK3. [score:7]
Consistently, Klf4 upregulation was detected when miR-7a was blocked in J1 mESCs using specific antisense inhibitors (Fig 6D). [score:6]
In this study, we identified transcription factor Klf4 as a downstream target of CHIR, whose expression and functions are regulated by miR-7a and the Wnt/ β-catenin signaling pathway. [score:6]
However, overexpression of β-catenin could not repress miR-7a expression, suggesting a β-catenin-independent manner between CHIR signaling and Klf4 regulation mediated by miR-7a. [score:6]
Thus, we further investigated miRNAs that potentially target the 3′-UTR of Klf4 and found that miR-7a could inhibit Klf4 expression. [score:5]
S4 Fig MiR-7a expression vector pCDH-mir-7a and their negative control pCDH-GFP were transfected into J1 mESCs, and miR-7a expression was detected by RT-qPCR. [score:5]
We also found that CHIR inhibited miR-7a expression (Fig 6E). [score:5]
We found that only miR-7a significantly suppressed Klf4 expression (Fig 6B and S4 Fig). [score:5]
However, overexpression of β-catenin was unable to repress miR-7a expression (Fig 6F). [score:5]
Klf4 is directly targeted by miR-7a. [score:4]
Thus, these results suggest that CHIR regulates Klf4 expression mediated by miR-7a in a β-catenin-independent manner. [score:4]
In this study, we showed that CHIR enhanced Klf4 expression through β-catenin signaling and miR-7a regulation in J1 mESCs. [score:4]
We performed an RT-qPCR assay and found that CHIR inhibited miR-7a expression (Fig 6E), while BIO was used as a positive control. [score:4]
In the lower panel, psiCHECK2- Klf4-3′UTR or psiCHECK2 control plasmids were co -transfected with mimics NC or miR-7a mimics/inhibitor into 293FT cells. [score:3]
As shown in Fig 6C, the reporter harboring the 3′-UTR fragment of Klf4 was significantly repressed, whereas miR-7a inhibitor rescued luciferase activity. [score:3]
To generate a miR-7a expression vector, a fragment carrying pre-miR-7a was amplified from genomic DNA of J1 ESCs and cloned into the EcoRI and NotI sites of the pCDH-CMV-MCS-EF1-coGFP vector (System Biosciences, Mountain View, CA, USA). [score:3]
The expression level of miR-7a. [score:3]
J1 mESCs were transfected with miR-7a expression vector pCDH-mir-7a and the negative control pCDH-GFP. [score:3]
At 48 h of incubation, miR-7a expression level was analyzed by RT-qPCR. [score:3]
J1 mESCs were transfected with mimics NC or miR-7a mimics/inhibitor. [score:3]
J1 mESCs were cultured in LIF-containing medium with or without CHIR/BIO for 24 h, and miR-7a expression level was analyzed by RT-qPCR. [score:3]
Presentation of miR-7a, miR-125, miR-152, and miR-363 target sites in the 3′-UTR of Klf4. [score:3]
Furthermore, transfection of miR-7a mimics suppressed Klf4 mRNA levels in J1 mESCs, as analyzed by RT-qPCR. [score:3]
Thus, these results indicate together with previous reports [31, 32] that miR-7a represses Klf4 expression. [score:3]
These results demonstrated that miR-7a may be a potential miRNA that represses the expression of Klf4; therefore, we selected miR-7a for further investigation. [score:1]
To further confirm that miR-7a was functional for Klf4, we subcloned the 3′-UTR fragment of Klf4 downstream the reporter gene into the psiCHECK-2 vector (Fig 6C, upper panel). [score:1]
were performed by co-transfecting the reporter vector and miR-7a mimics into 293FT cells. [score:1]
The 3′-UTR of Klf4 that we used, contains two putative miR-7a binding sites, consistent with previous studies [31, 32]. [score:1]
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15
[+] score: 80
Other miRNAs from this paper: mmu-mir-7a-1, mmu-mir-7b
The results showed that miR-7 expression level was significantly higher in Non-tumor normal mucosa (P<0.01), which suggested miR-7 might play tumor suppressor role and its activity was inhibited in gastric tumors. [score:7]
COX-2 was a functional target of miR-7. COX-2 is the direct target of miR-7.. [score:6]
The activation of miR-7 and the suppression of COX-2 in the Canolol -treated Tg group were observed simultaneously in our research, and COX-2 was showed a functional target of miR-7 (Fig. 7). [score:5]
According to the prediction of miR-7 target using TargetScan (http://www. [score:5]
To elucidate the underlying mechanisms of the suppressive effects of miR-7 on tumor progression, TargetScan (http://www. [score:5]
org/) were used to predict the miR-7 targets, and COX-2, a pivotal factor in the COX-2/PGE2 signal pathway, was found to be the possible target. [score:5]
Briefly, miR-7 was silenced expressed in tumor tissue in Non-treatment Tg mice, while the miR-7 expression was reactivated after canolol administration (Fig. 6B). [score:5]
The results suggested that miR-7 downregulated the gene transcription of COX-2 through the incomplete combination with 3’-UTR region, and interrupted the PGE2 synthesis, and then blocked the whole signaling pathway. [score:4]
In addition, miR-7 was found downregulated in gastric tumors in an inflammation -dependent manner in gastric cancer[16]. [score:4]
Accumulating evidences showed miRNAs played essential roles in tumor initiation, progression and metastasis, and miR-7, one of the inflammation-related miRNAs in gastric tumor, was delineated to be a potential tumor suppressor[16]. [score:3]
In addition, the miR-7 up eukaryotic expression plasmid and the mutants of COX-2 3’-UTR plasmids were also supplied by Shanghai GeneChem. [score:3]
Expression of COX-2/PGE2 signal pathway genes and miR-7 in gastric tumor. [score:3]
To further explore the mechanism of anti-inflammation and anti-tumor effects of canolol, the miR-7 expression was detected using qRT-PCR. [score:3]
However, in the Tg mice receiving canolol administration, the miR-7 expression was reactivated significantly (P<0.01) (Fig. 6B). [score:3]
As the analysis of miR-7 expression, the Ct is the fractional cycle number at which the fluorescence of each sample passes the fixed threshold. [score:3]
To further study whether miRNAs participate in the COX-2/PGE2 signal transduction, the relative expression level of miR-7 was analyzed using qRT-PCR. [score:3]
The effects of canolol on the COX-2/PGE2 pathway genes (A) and miR-7 expression (B). [score:3]
The expression of miR-7 and the other genes was assayed on ABI7500 real-time PCR detection system and normalized with snoRNA202 and GAPDH respectively. [score:2]
We found that miR-7 inhibited the luciferase activity of a reporter containing the wild-type COX-2 3’-UTR-1 but not that of 3’-UTR-2 after the dual-luciferase reporter assay (Fig. 7A). [score:2]
In addition, two regions, position 86–92 (3’-UTR-1) (Fig. 7B) and 1111–1118 (3’-UTR-2) (Fig. 7C) of COX-2 have putative miR-7 -binding elements. [score:1]
miR-7 has been proven to play a substantial role in hepatocellular carcinoma [13], breast cancer[14], and lung cancer[15]. [score:1]
org/), two regions, position 86–92 and 1111–1118 of COX-2 were combined with miR-7 seed region possibly. [score:1]
miR-7 and its putative two binding sequence in the 3’-UTR of COX-2, 86–92 sites (B) and 1111–1118 sites (C). [score:1]
HEK239T cells in logarithmic growth phase were seeded in 24-well plate, and were cotransfected with 1μg miR-7 and COX-2 plasmid using lipo 2000 (Invitrogen) as the manual protocol. [score:1]
However, a functional link between miR-7 and the COX-2/PGE2 pathway has not been established. [score:1]
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[+] score: 54
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-24-1, hsa-mir-24-2, hsa-mir-25, mmu-let-7g, mmu-let-7i, mmu-mir-124-3, mmu-mir-9-2, mmu-mir-134, mmu-mir-137, mmu-mir-138-2, mmu-mir-145a, mmu-mir-24-1, hsa-mir-192, mmu-mir-194-1, mmu-mir-200b, hsa-mir-7-1, hsa-mir-7-2, hsa-mir-7-3, hsa-mir-215, hsa-mir-221, hsa-mir-200b, mmu-mir-296, mmu-let-7d, mmu-mir-106b, hsa-let-7g, hsa-let-7i, hsa-mir-124-1, hsa-mir-124-2, hsa-mir-124-3, hsa-mir-137, hsa-mir-138-2, hsa-mir-145, hsa-mir-9-1, hsa-mir-9-2, hsa-mir-9-3, hsa-mir-134, hsa-mir-138-1, hsa-mir-194-1, mmu-mir-192, 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-24-2, mmu-mir-346, hsa-mir-200c, mmu-mir-17, mmu-mir-25, mmu-mir-200c, mmu-mir-221, mmu-mir-124-1, mmu-mir-124-2, mmu-mir-9-1, mmu-mir-9-3, mmu-mir-138-1, mmu-mir-7a-1, mmu-mir-7b, hsa-mir-194-2, mmu-mir-194-2, hsa-mir-106b, hsa-mir-200a, hsa-mir-296, hsa-mir-369, hsa-mir-346, mmu-mir-215, gga-let-7i, gga-let-7a-3, gga-let-7b, gga-let-7c, gga-mir-221, gga-mir-17, gga-mir-138-1, gga-mir-124a, gga-mir-194, gga-mir-215, gga-mir-137, gga-mir-7-2, gga-mir-138-2, gga-let-7g, gga-let-7d, gga-let-7f, gga-let-7a-1, gga-mir-200a, gga-mir-200b, gga-mir-124b, gga-let-7a-2, gga-let-7j, gga-let-7k, gga-mir-7-3, gga-mir-7-1, gga-mir-24, gga-mir-7b, gga-mir-9-2, dre-mir-7b, dre-mir-7a-1, dre-mir-7a-2, dre-mir-192, dre-mir-221, dre-mir-430a-1, dre-mir-430b-1, dre-mir-430c-1, 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-7c-1, dre-let-7c-2, dre-let-7d-1, dre-let-7d-2, dre-let-7e, dre-let-7f, dre-let-7g-1, dre-let-7g-2, dre-let-7h, dre-let-7i, dre-mir-7a-3, dre-mir-9-1, dre-mir-9-2, dre-mir-9-4, dre-mir-9-3, dre-mir-9-5, dre-mir-9-6, dre-mir-9-7, dre-mir-17a-1, dre-mir-17a-2, dre-mir-24-4, dre-mir-24-2, dre-mir-24-3, dre-mir-24-1, dre-mir-25, dre-mir-92b, dre-mir-124-1, dre-mir-124-2, dre-mir-124-3, dre-mir-124-4, dre-mir-124-5, dre-mir-124-6, dre-mir-137-1, dre-mir-137-2, dre-mir-138-1, dre-mir-145, dre-mir-194a, dre-mir-194b, dre-mir-200a, dre-mir-200b, dre-mir-200c, dre-mir-430c-2, dre-mir-430c-3, dre-mir-430c-4, dre-mir-430c-5, dre-mir-430c-6, dre-mir-430c-7, dre-mir-430c-8, dre-mir-430c-9, dre-mir-430c-10, dre-mir-430c-11, dre-mir-430c-12, dre-mir-430c-13, dre-mir-430c-14, dre-mir-430c-15, dre-mir-430c-16, dre-mir-430c-17, dre-mir-430c-18, dre-mir-430a-2, dre-mir-430a-3, dre-mir-430a-4, dre-mir-430a-5, dre-mir-430a-6, dre-mir-430a-7, dre-mir-430a-8, dre-mir-430a-9, dre-mir-430a-10, dre-mir-430a-11, dre-mir-430a-12, dre-mir-430a-13, dre-mir-430a-14, dre-mir-430a-15, dre-mir-430a-16, dre-mir-430a-17, dre-mir-430a-18, dre-mir-430i-1, dre-mir-430i-2, dre-mir-430i-3, dre-mir-430b-2, dre-mir-430b-3, dre-mir-430b-4, dre-mir-430b-6, dre-mir-430b-7, dre-mir-430b-8, dre-mir-430b-9, dre-mir-430b-10, dre-mir-430b-11, dre-mir-430b-12, dre-mir-430b-13, dre-mir-430b-14, dre-mir-430b-15, dre-mir-430b-16, dre-mir-430b-17, dre-mir-430b-18, dre-mir-430b-5, dre-mir-430b-19, dre-mir-430b-20, mmu-mir-470, hsa-mir-485, hsa-mir-496, dre-let-7j, mmu-mir-485, mmu-mir-543, mmu-mir-369, hsa-mir-92b, gga-mir-9-1, hsa-mir-671, mmu-mir-671, mmu-mir-496a, mmu-mir-92b, hsa-mir-543, gga-mir-124a-2, mmu-mir-145b, mmu-let-7j, mmu-mir-496b, mmu-let-7k, gga-mir-124c, gga-mir-9-3, gga-mir-145, dre-mir-138-2, dre-mir-24b, gga-mir-9-4, mmu-mir-9b-2, mmu-mir-124b, mmu-mir-9b-1, mmu-mir-9b-3, gga-mir-9b-1, gga-let-7l-1, gga-let-7l-2, gga-mir-9b-2
However, more functional analysis of naturally expressed circRNAs within the CNS will provide useful information regarding the precise role of circRNAs and other long-ncRNAs in the regulation of gene expression required through the different developmental stages of the CNS and also, whether miRNAs other than miR-7 are regulated by long-ncRNAs. [score:8]
This observation suggest that miR-671 might function as an indirect regulator of miR-7 activity by targeting and reducing ciRS-7 levels; however, the exact function of the ciRS-7:miR-671 interaction during the development of the CNS is still unknown. [score:6]
Moreover, due to the high degree of conservation of miR-7, the binding sites in the human CDR1as are functional when it is expressed in zebrafish resulting in impaired midbrain development which is similar to the phenotype of knocking-down miR-7 (Memczak et al., 2013). [score:5]
Interestingly, the concentration gradient of a single miRNA is capable of determining specific zones of neuronal differentiation as it is the case of miR-7 that maintains the proper localization of dopaminergic neuronal differentiation regions within the mouse olfactory bulb by having an opposite concentration/expression gradient to that of its target gene, Pax6 (De Chevigny et al., 2012). [score:5]
Silencing QKI in the U343 glioblastoma cell line, results in miR-7 expression and cell cycle arrest, through a mechanism involving miR-7 negative regulation of epidermal growth factor (EGF) receptor (EGFR) protein levels, thus blunting the EGF -dependent ERK activation (Wang et al., 2013). [score:4]
In particular, the human circRNA antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as) contains 63 conserved binding sites for miR-7 and specifically regulates miR-7 expression in neuronal tissues (Memczak et al., 2013). [score:4]
ciRS-7 contains more than 70 conserved binding sites for miR-7 and when miR-7 binds to it, AGO is recruited and binds to ciRS-7:miR-7 complexes however, ciRS-7 is resistant to miR-7 -mediated destabilization resulting in miR-7 activity blockage and derepression of miR-7 target genes (Hansen et al., 2013). [score:3]
Moreover, knock-out mice for MSI2 present higher levels of mature miR-7 without a change in pri-miR-7 abundance confirming that RBPs are key players in the regulatory mechanism controlling miRNA biogenesis (Choudhury et al., 2013). [score:3]
Locked nucleic acid -based in situ hybridisation reveals miR-7a as a hypothalamus-enriched microRNA with a distinct expression pattern. [score:3]
A recent report, demonstrated that the processing of the miR-7 pre-miRNA generated from the heterogeneous nuclear ribonucleoprotein K (hnRNP K) pre-mRNA transcript, is inhibited in non-brain human and mouse cells due to the binding of the RNA binding proteins (RBPs) Musashi homolog 2 (MSI2) and Hu antigen R (HuR) to the terminal loop of the pri-miR-7 and the stabilization of the pri-miRNA structure (Choudhury et al., 2013). [score:3]
Absence of QKI-5 and QKI-6 results in increased mature miR-7 levels due to the fact that these proteins negatively regulate pri-miR-7 to miR-7 processing by maintaining the pri-miR-7 at the nucleus and tightly bounded by Drosha (Wang et al., 2013). [score:2]
In addition, miR-7 biogenesis regulation also occurs via MSI2 and HuR binding during the in vitro neuronal differentiation of the SH-SY5Y cell line (Choudhury et al., 2013). [score:2]
miR-7a regulation of Pax6 controls spatial origin of forebrain dopaminergic neurons. [score:2]
Another study showed that the control of miR-7 biogenesis by the quaking (QKI) RBPs, isoforms QKI-5 and QKI-6 that are localized at the nucleus and throughout the cell respectively, contribute to regulate the proliferation rate of glioblastoma cells cultures (Wang et al., 2013). [score:2]
An independent study, described ciRS-7, another circRNA, as a miR-7 sponge in the human brain and in mouse neocortical and hippocampal neurons (Hansen et al., 2013). [score:1]
Tissue-specific control of brain-enriched miR-7 biogenesis. [score:1]
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[+] score: 44
cryano (linc-oip5) is a long intergenic ncRNA, first identified in Zebrafish has been reported to be overexpressed in morula stage of the mouse embryo and maintains stem cell self-renewal in embryo through modulation of NANOG by sponging Nanog targeting miR-7a 16, 17. [score:5]
OIP5-AS1, a long intergenic ncRNA transcribed in antisense from the OIP5 gene, located at chromosome 15q15.1, was first discovered as cyrano in zebrafish and reported to function as a major regulator of neurogenesis during development and in the maintenance of self-renewal in mouse morula stage modulating the nanog expression by sponging mmu-miR-7 16, 17. [score:5]
Human NANOG is not post-transcriptionally regulated by OIP5-AS1In the mouse embryo, overexpression of Oip5-as1 (1700020I14Rik) was shown to maintain stemness by regulating the steady state level of NANOG through sponging of mmu-miR-7a 16– 18. [score:5]
In the mouse embryo, overexpression of Oip5-as1 (1700020I14Rik) was shown to maintain stemness by regulating the steady state level of NANOG through sponging of mmu-miR-7a 16– 18. [score:4]
OIP5-AS1 can sponge miRNAs with tumor suppressive functionSince human NANOG is not regulated by hsa-miR-7a-5p due to lack of binding sites, we asked the question whether the OIP5-AS1 could maintain the cellular stemness by sponging other miRNAs that can post-transcriptionally control stemness associated TFs factors. [score:4]
Overexpressed Oip5-as1 maintains stemness by sponging the mmu-miR-7a-5p [16]. [score:3]
To the surprise, the 3′-UTR of human NANOG did not carry any binding site for hsa-miR-7-5p suggesting that NANOG was not targeted by hsa-miR-7-5p. [score:3]
In mouse, overexpressed Oip5-as1 sponge the cell differentiation promoting mmu-miR-7a-5p and maintains stemness modulating the nanog level. [score:3]
Cui Y The miR-7 identified from collagen biomaterial -based three-dimensional cultured cells regulates neural stem cell differentiationStem Cells Dev. [score:2]
Since human NANOG is not regulated by hsa-miR-7a-5p due to lack of binding sites, we asked the question whether the OIP5-AS1 could maintain the cellular stemness by sponging other miRNAs that can post-transcriptionally control stemness associated TFs factors. [score:2]
In mouse, mmu-miR-7a-5p binds to the 3′-UTR of Nanog, regulates its steady state level and signals for cellular differentiation. [score:2]
However, the binding site for miRNA miR-7 was conserved in OIP5-AS1 (Fig.   2b and Supplementary Fig.   S1). [score:1]
Since the hsa-miR-7a mature sequence is 100% conserved, we checked whether a similar mechanism is taking place in humans. [score:1]
Therefore, we checked the binding sites for the mouse miRNA counterpart in human (hsa-miR-7-5p), as this particular miRNA is highly conserved across the vertebrates [17]. [score:1]
Though human OIP5-AS1 was poorly conserved, the binding site for hsa-miR-7-5p is preserved at a conserved part of the transcript. [score:1]
Therefore, we checked the conservation of hsa-miR-7–5p and mmu-miR-7 mature sequence and observed 100% sequence similarity between mouse and human (Fig.   2a). [score:1]
We did a clustal alignment of mouse Nanog and human NANOG mRNA and found that human NANOG 3′-UTR is not conserved and thus no binding site for hsa-miR-7 (Supplementary Table  S1). [score:1]
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[+] score: 32
In neurons, miR-7 was identified as a regulator of alpha-synuclein, whose over -expression is associated with impaired function of dopamine-generating cells and development of Parkinson's disease. [score:7]
In the study by Hansen et al. (2011), the circRNA CDR1as, which is the antisense transcript of CDR1, has been identified to be targeted by miR-7, a miRNA implicated in various disease, including Parkinson's disease and several types of cancers. [score:7]
Its expression was primarily found in the brain where it was co-expressed with miR-7 (Hansen et al., 2011). [score:5]
The study by Hansen et al. (2011) revealed overlaps in expression of the circRNA CDR1as and miR-7 in mouse brain suggesting that a major amount of miR-7 is attached to CDR1as in mouse brain. [score:3]
Repression of alpha-synuclein expression and toxicity by microRNA-7. Proc. [score:3]
The fact that circular RNAs are targeted by endogenous miRNAs was reported by Hansen et al. The authors outlined a circRNA destruction mechanism in which miR-671 binds CDR1as/ciRS-7 with greater complementarities than miR-7 and induces cleavage of this circRNA mediated by Ago [the catalytic component of RNA induced silencing complex (RISC) which is a central component of RNA interference (RNAi) machinery] (Hansen et al., 2011). [score:3]
Loss of miR-7 in cultured Parkinson's disease cells possibly contributes to increased alpha-synuclein level (Junn et al., 2009). [score:3]
Circular RNA and miR-7 in cancer. [score:1]
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[+] score: 30
Interestingly, by searching candidate microRNAs that bind to the human EGFR 3′-UTR using TargetScan 5.1, miRanda, miRbase and PITA software programs and our recent microRNA expression profiling analysis [6], we came up with 2 microRNAs, miR7 and miR141, that were predicted to be able to bind to the human EGFR 3′-UTR (Figure 3A) and that both of the microRNAs are downregulated in KLF8-overepressed cells [33]. [score:8]
Figure 3 A. KLF8 inhibits the expression of miR7, miR141 and miR200c. [score:5]
A. KLF8 inhibits the expression of miR7, miR141 and miR200c. [score:5]
miR7 has been reported to inhibit EGFR [34]. [score:3]
We also identified miR7 as a target of repression by KLF8 [6] (Figure 3). [score:3]
To test the potential role of miR7 and miR141 on EGFR expression in our experimental system, we first confirmed the microRNA array data by qPCR (Figure 3A). [score:3]
Although modestly repressed by KLF8, miR7 may also play a partial role in the regulation of EGFR expression downstream of KLF8, which is worth of further investigation. [score:2]
miR7 has been reported to repress EGFR in glioblastoma [34]. [score:1]
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[+] score: 26
microRNA-7 down-regulation mediates excessive collagen expression in localized scleroderma. [score:6]
microRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway and is down-regulated in glioblastoma. [score:6]
An inverse effect has intriguingly been observed for miR-7a, which predictably targets VAChT and AChE-R and may hence reduce the packaging efficiency of ACh and limit cholinergic signals: miR-7a alleviates the maintenance of neuropathic pain through regulating neuronal excitability by targeting the β 2 subunit of the voltage -dependent sodium channel (Sakai et al., 2013). [score:6]
Repression of alpha-synuclein expression and toxicity by microRNA-7. Proc. [score:3]
Decreased miR-7 expression in the skin and sera of patients with dermatomyositis. [score:3]
miR-7a alleviates the maintenance of neuropathic pain through regulation of neuronal excitability. [score:2]
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[+] score: 25
A circRNA from the Zfyve9 gene (mm9_circ_014815) that was age -upregulated in hippocampus harbored several different microRNA target sites: 3 target sites for miR-9, a microRNA with roles in neural development and neural pathologies 32, 1 target site for miR-124, a highly abundant brain miRNA that is implicated in central nervous system disorders 33, and 1 miR-7 target site (Supplementary Table S13). [score:13]
For instance, CDR1as, the miR-7 sponging circRNA 3 11, harbored 36 highly conserved miR-7 target sites in our analysis and was confirmed to be upregulated during aging in the cortex (Fig. 3c &0). [score:6]
Three target sites for miR-7 were also found in exons of a circRNA from the Hecw1 locus (mmu_circ_0004501), which was upregulated in both old cortex and hippocampus (Supplementary Tables S10 and S13). [score:6]
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[+] score: 23
In an intriguing elaboration of this regulatory pathway, the activity of the mammalian miR-7 miRNA can be inhibited by CDR1as/ciRS-7, which is in turn targeted by another miRNA, miR-671, which shows near-perfect complementarity and triggers endonucleolytic cleavage of CDR1as [8– 10]. [score:6]
Based on several criteria, including their intriguing expression patterns, their apparently elevated sequence conservation and the compelling hypothesis that CDR1as acts as a miR-7 sponge, these circRNAs have been proposed to comprise a large class of post-transcriptional regulators. [score:4]
With >60 conserved sites for miR-7, CDR1as is thought to act as a sponge to titrate miR-7 from its other targets [8, 9]. [score:3]
Although a biological context has not yet been identified in which CDR1as loss-of-function influences miR-7 activity, this circRNA has >60 conserved sites to miR-7 and a developmental phenotype following its ectopic delivery [8, 9]. [score:2]
Strikingly, when counting the clusters of AGO2 crosslinks mapping to each circRNA [27], CDR1as had 26 clusters corresponding to miR-7 sites, which was by far the most mapping to any circRNA for any miRNA family (Figure  6B). [score:1]
The circRNA with the most compelling evidence for a biological function is the miR-7 sponge, CDR1as. [score:1]
CDR1as-miR-7 was also the only circRNA-miRNA pair that exceeded the upper limit of results from the negative control, in which the analysis was repeated with permutated miRNA sequences (Figure  6C). [score:1]
CDR1as also had 22 sites for the miR-876-5p/3167 family (Figure S9B in Additional file 12), although they were not as conserved as the miR-7 sites. [score:1]
The previously proposed miR-7 sponge, CDR1as, is one of only two circRNAs with more miRNA sites than expected by chance, with the next best miRNA-sponge candidate deriving from a gene encoding a primate-specific zinc-finger protein, ZNF91. [score:1]
Again, CDR1as ranked on top, containing 71 miR-7 sites (Figure  6C). [score:1]
The outlying CDR1as-miR-7 pair is indicated. [score:1]
Mammalian cells produce a large number of circRNAs, which have captured the interest of many biologists, particularly after the description of CDR1as and its many conserved sites to miR-7. Our work identifies thousands of additional circRNAs and focuses on those that have circular fractions ≥10%. [score:1]
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[+] score: 18
Expression of miR-124 and miR-137, respectively, increased up to 8- and 24-fold, expression of miR-129 and miR-139, respectively, decreased up to 2- and 4-fold, and expression of miR-7 and miR-218 did not change appreciably. [score:7]
We identified six miRNAs of particular interest, miR-7, miR-124, miR-129, miR-137, miR-139 and miR-218, which were down-regulated in both AAs and GBMs (Figure 1A, Additional file 8 and Table 1) at a more stringent level of significance (P ≤ 0.01). [score:4]
Of the 35 miRNAs, we identified six HGA-miRNAs, which were down-regulated in both AA and GBM tumors at a more stringent degree of significance (P < 0.01): miR-7, miR-124, miR-129, miR-137, miR-139 and miR-218. [score:4]
We observed that the majority of the HGA-miRNAs show expression changes during, or have been implicated in, differentiation of various cell lineages: miR-7 during photoreceptor differentiation [23]; miR-124 and miR-137 during erythropoiesis [24]; miR-124 and miR-218 during neuronal differentiation of embryonal carcinoma cell differentiation [25]; miR-124 during neuronal differentiation of ES cells [12]. [score:3]
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24
[+] score: 17
For example, miR-7, -433 and -153 bind to the 3′-untranslated region of α-synuclein to inhibit the gene expression in DA neurons [23, 24]. [score:7]
For example, miR-7 regulates the expression of A53T α-synuclein [51], miR-7 and -153 participate in SNCA transcription [23, 51], the function of miR-1224, -184 and let-7i-3p/5p is regulated by leucine-rich repeat kinase 2 (LRRK2) [52, 53], and miR-127-5p and -16-5p play their roles in glucocerebrosidase (GBA) pathway [54]. [score:5]
In this sequencing, miR-9-5p and miR-7a-5p were most abundantly expressed in the A53T mice [Transcripts per kilobase million (TPM) = 153248.90 and 97029.90, respectively], but the expression was not changed dramatically compared with WT mice (fold-changes of 0.90 and 1.15, respectively). [score:4]
And some PD-related miRNAs, included miR-7, -153, -1224, -184 and let-7i-3p, were just changed slightly in our results [23, 51– 53]. [score:1]
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25
[+] score: 16
We performed microarray analysis of miRNA expression of glioma cells silenced for TALNEC2 and found that silencing of TALNEC2 increased the expression of several tumor suppressor miRNAs such as miR-137, miR-124, miR-205, miR-7 and miR-492, whereas it decreased the expression of some oncomiRs such as miR-21, miR-155, miR-33b and miR-191. [score:9]
We found that silencing of TALNEC2 in U87 cells resulted in an increased expression of miRNAs associated with tumor suppression [38, 39] (e. g., let-7b, miR-7, miR-124, miR-137, miR-129-3p, miR-142-3p, miR-205, miR-376c, miR-492, miR-562 and miR-3144) and in a decrease in the expression of miRNAs associated with tumor promotion [38– 40] (e. g., miR-9, miR-21 miR-33b, miR-155, miR-191, miR-525-3p, and miR-767-3p). [score:7]
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[+] score: 16
The 4.8-kb gigaloop is a putative structure formed by pairing of VCR and a complementary sequence (cVCR) Figure 2. Conserved sequences of stratum 1 (shared by Homo and Callorhinchus IGF1R 3'-UTRs): (a) the 3' end of the long IGF1R transcript; (b) a miR-7-3p target site that has been lost from the Pelodiscus sequence; (c) let-7-3p target site; (d) miR-186 target site; (e) The VCR with predicted binding sites for miR-376c, miR-675 (derived from the imprinted H19 RNA) and miR-16. [score:7]
In addition to the polyadenylation site, this region contains a miR-7-3p target site that has been lost from the Pelodiscus sequence (Fig.  2b), a conserved let-7-3p target site (Fig.  2c) and a conserved miR-186 target site (Fig.  2d). [score:7]
Postranscriptional regulation of IGF1R by let-7, miR-7 and miR-182 are plausible candidates for such ancient functions because these microRNA families are themselves ancient, conserved between protostomes and deuterostomes [32–34]. [score:2]
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27
[+] score: 15
Downregulation of miR-7 was previously reported in glioblastoma [24] along with underexpression of miR-128 in lung cancer [23], but in HNSCC cell lines the expression of these miRNAs was variable as compared to controls (Fig. S1A). [score:7]
Given that miR-7 has known interactions with EGFR as both a tumor suppressor and oncogene [25, 29], but did not show an effect on cell viability in HNSCC, we postulated that miR-27a* targets additional genes in the EGFR signaling axis to reduce cell survival. [score:5]
We used software prediction programs to identify candidate binding sites of miRNAs within the EGFR gene and identified miR-7, −27a (miR-27a-3p), −27a*, −27b (miR-27b-3p), −27b* (miR-27b-5p), and −128 (Fig. 1A). [score:1]
Evaluation of miR-7 expression did not demonstrate a significant difference in the entire cohort or the matched samples. [score:1]
To assess the effects of the miRNAs on tumor viability, three HNSCC cell lines were transfected with miR-7, −27a and −27a* mimics. [score:1]
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However, the expression dynamics of miR-34c, let-7a, miR-7a were different; the expression level was increased with increase in size of follicles. [score:5]
a) 6d-8d, b) 8d-12d, c) 12d-15d, d) 15d-21d, e) 21d-P6, f) P48-h6 To further validate these differentially expressed miRNAs identified from the mouse ovary, the expression levels of miR-199a, miR-470, miR-871, miR-34c let-7a, miR-7a, miR-351, miR-191 were further examined in different size follicles (i. e., 100 μm −130 μm, 200 μm -280 μm, 450 μm -550 μm, 500 μm -600 μm) using qRT-PCR assay. [score:4]
h) Expression profile of miR-7a through qRT-PCR Sequencing data was subjected to Rfam to filter out rRNAs, tRNAs, snRNAs and snoRNAs. [score:3]
H) Expression profile of mmu-mir-7a in sequencing data. [score:3]
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29
[+] score: 14
To determine whether there are direct interactions between miR-2, miR-133, miR-7, miR-79 and their target sites of the crab cyclin B gene, we used a luciferase 3′-UTR reporter assay to measure the inhibitory effects of these miRNAs. [score:3]
As shown in Figure  2, the 5′ seed sequences of miR-2, miR-7 miR-79 and miR-133 were revealed to be complementary to their corresponding target sites such as GY-box, Brd-box, and K-box in 3′-UTR of the crab cyclin B gene. [score:3]
To identify miRNAs differentially expressed during the crab oocyte maturation, the relative abundance of miR-2, miR-7, miR-79, miR-133 and other six selected miRNAs in the ovaries at GV and MI stages was assessed by quantitative real-time PCR. [score:3]
Figure 2 The potential miRNA target sites of miR-2, miR-7, miR-79 and miR-133 in the 3′-UTR of the crab cyclin B as detected by RNAhybrid [19]. [score:3]
The 5′ seed sequences of four miRNAs, miR-2, miR-7, miR-79 and miR-133, were revealed to complementary to miRNA binding sites in 3′-UTR of the cyclin B. Quantitative real time PCR analysis showed that miR-2 and miR-133 are much more abundant in the first metaphase (MI) of meiosis than in germinal vesicle (GV) stage. [score:1]
After cotransfected HEK 293 T cells, miR-2 and miR-133 mimics significantly reduce the luciferase activity from the reporter construct containing the cyclin B 3′-UTR, whereas miR-7, miR-79 and negative control mimics have no effect on the luciferase activity (Figure  5A). [score:1]
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[+] score: 13
To test this possibility, we used the miRNA target-prediction program, TargetScan, to screen for miRNA -binding seed sequences in the Olfr mRNAs we defined as well as the data set from Ibarra-Soria et al. In particular, we used this in silico screen to examine the following miRNAs: miR-200a/miR-141a, the only miRNA known to have a role in olfactory neurogenesis (53), and eight well-studied neurally expressed miRNAs: miR-7, -9, -124, -128, -132, -134, -138 and let-7 (38, 54– 61). [score:7]
Chen H. Shalom-Feuerstein R. Riley J. Zhang S. -D. Tucci P. Agostini M. Aberdam D. Knight R. A. Genchi G. Nicotera P. miR-7 and miR-214 are specifically expressed during neuroblastoma differentiation, cortical development and embryonic stem cells differentiation, and control neurite outgrowth in vitro Biochem. [score:4]
During the course of this analysis, we noted that a relatively large percentage of Gpcr genes (>15%) contain predicted miR-7 and -128 binding sites (Figure 3C), suggesting that miR-7, -200a, -9 and -128, in particular, may be broad regulators of Gpcr mRNAs. [score:2]
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[+] score: 13
In a more recent study, the HBV-encoded x protein indirectly downregulated EGFR expression in HCC cells via microRNA-7, resulting in decreased growth rate of these cells. [score:7]
Chen Y. J. Chien P. H. Chen W. S. Chien Y. F. Hsu Y. Y. Wang L. Y. Chen J. Y. Lin C. W. Huang T. C. Yu Y. L. Hepatitis B virus-encoded x protein downregulates EGFR expression via inducing microRNA-7 in hepatocellular carcinoma cells Evid. [score:6]
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miR-26a, miR-34a and miR-146a suppress HCC cell progression by targeting fucosyltransferase 8 (FUT8), the only enzyme responsible for β1,6-fucosylation of N-glycans [14]; O-GlcNAc transferase (OGT) was identified as a novel target of miRNA-7 in a mouse glioblastoma xenograft mo del [15] and of miR-24-1 in human breast cancer cells [16]; mature miR-17-5p and passenger miR-17-3p induce HCC by targeting N-acetylgalactosaminyltransferase 7 (GALNT7) [17]; Let-7c inhibits metastatic ability of mouse hepatocarcinoma cells via targeting mannoside acetylglucosaminyltransferase 4 isoenzyme A (Mgat4a) [11]. [score:13]
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[+] score: 13
miRNA (miR-7 and miR-34a) target gene (EGFR and AXL) expression in tissues. [score:5]
Therefore, we used the same RNA from the 5 extraction methods and evaluated the expression of validated targets of two of the miRNAs, namely EGFR and AXL, targets of miR-7 and miR-34a respectively (Fig.   5). [score:5]
We chose to look at six miRNAs (see Additional file  5: Figure S1) that differed in GC content (35–50%), secondary structure (ΔG = − 3.6 – 0.5) and were especially applicable to cancer studies given their tumour suppressive (miR-7-5p, miR-34a-5p) or oncogenic (miR-21-5p) functions, or their involvement in epithelial-mesenchymal transition (EMT, miR-200 family) [17– 23]. [score:3]
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miR-7, which represses the expression of Yan protein and promotes photoreceptor differentiation [44], as well as miR-125-2b, highly abundant in adult retina [45], were down-regulated over 48 hours post-ESMV treatment. [score:6]
The concurrent down-regulation of the miR-let7 cluster, known to promote differentiation in most cells and de-differentiation when it is inhibited [76], miR-125, a highly abundant miRNA in adult retina [45], and miR-7, known to promote photoreceptor differentiation [44] is consistent with Müller cell de-differentiation under the influence of ESMVs. [score:6]
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For instance, significant down-regulation of circRNA ITCH was discovered in lung cancer and up -regulating its expression could markedly elevate its parental cancer-suppressive gene ITCH through sponging oncogenic miR-7 and miR-214 [9]. [score:9]
Hansen et al. identified ciRS-7, a circular RNA, which acts as a designated miR-7 inhibitor/sponge, has conceptually changed the mechanistic understanding of miRNA networks in cancers [8]. [score:3]
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Several down-regulated (i. e. miR-1, miR-7, miR-34a, miR-122, miR-125b, miR-200) or up-regulated (i. e. miR-17, miR-18, miR-19, miR-155, miR-93, miR-221/222) miRNAs have been identified as tumor suppressor or oncomirs, respectively, by targeting and regulating genes involved in cell proliferation, apoptosis, angiogenesis and metastasis [13]. [score:12]
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[+] score: 12
Other miRNAs from this paper: mmu-mir-7a-1, mmu-mir-7b
Homeobox protein D10 is a target of the microRNA miR-7, which suppresses p21/CDKN1a (Cyclin -dependent kinase inhibitor 1α) activated kinase [53]. [score:7]
Reddy S. D. Ohshiro K. Rayala S. K. Kumar R. MicroRNA-7, a homeobox D10 target, inhibits p21-activated kinase 1 and regulates its functions Cancer Res. [score:5]
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38
[+] score: 11
Increasing studies indicate that miRs (miR-7 and miR-138) could suppress the epithelial-to-mesenchymal transition and metastasis of cancer stem cells by targeting focal adhesion kinase (FAK) expression [16, 17]. [score:7]
FAK has been verified as the target gene for several other miRs (miR-543 and miR-7) in cancer development [17, 31]. [score:4]
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[+] score: 11
In sporadic Alzheimer's disease (AD), the brain-specific circRNA ciRS-7, which contains sites that are capable of sponging miR-7, was found to be downregulated compared to age-matched control brains, likely by increasing the availability of miR-7 to brain cells in AD [13]. [score:5]
ciRS-7 was also implicated in tumorigenesis, as sequestration of miR-7 by ciRS-7 would be expected to promote the expression of the oncogenic factors EGFR and XIAP [17]. [score:3]
Specific examples illustrating these functions include the exon–intron circRNAs EIciEIF3J and EIciPAIP2, which were found to promote gene transcription [9], and cir-ITCH, which sponged three microRNAs (miR-7, miR-17, and miR-214) and thereby increased the levels of ITCH, an inhibitor of the Wnt/β-catenin signaling pathway [16]. [score:3]
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[+] score: 11
Other miRNAs from this paper: mmu-mir-148a, mmu-mir-7a-1, mmu-mir-7b
Decreased levels of PTEN also concur with the upregulation of microRNA (miR-7) that downregulates PTEN expression. [score:9]
These findings suggest that defective miR-7 regulation of PTEN could contribute to B cell hyperresponsiveness in SLE [31]. [score:2]
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[+] score: 11
Knocking-down of Spl expression by small inhibitory RNA led to decreased IGFIR expression and attenuated growth and metastasis of gastric cancer cells [30], otherwise, IGF1R can be downregulated by miR-7 and significantly reduced GC cell migration and invasion [31]. [score:11]
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It was further demonstrated that the upregulation of miRNA7 expression in COPD patients was linked to the downregulation of Epac1 [106]. [score:9]
Oldenburger A. van Basten B. Kooistra W. Meurs H. Maarsingh H. Krenning G. Timens W. Schmidt M. Interaction between Epac1 and miRNA-7 in airway smooth muscle cellsNaunyn Schmiedebergs Arch. [score:1]
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43
[+] score: 10
Interestingly, 7 miRNAs (let-7k, miR-378d, miR-129-1-3p, miR-21a-5p, miR-29c-3p, miR-203-3p, and miR-7a-5p) expression was significantly restored after AOE treatment. [score:3]
The expression of a miR-let-7k, b miR-129-1-3p, c miR-378d, d miR-21a-5p, e miR-29c-3p, f miR-203-3p, g miR-7a-5p in DB/DB groups (white column), db-/db-H [2]O (gray column) and db-/db-AOE (black column) detected by QRT-PCR consist with sequencing. [score:3]
Moreover, there is also the first demonstrated elevated levels of miR-874-3p, miR-7a-5p, miR-455-5p, miR-129-1-3p, miR-151-5p, miR-3473b, and down regulated levels of miR-345-3p, novel_mir_8 and let-7 k in the kidneys of db-/db- mice. [score:2]
7 miRNAs (mmu-let-7k, mmu-miR-378d, mmu-miR-129-1-3p, mmu-miR-21a-5p, mmu-miR-29c-3p, mmu-miR-203-3p, and mmu-miR-7a-5p) were selected as candidate and quantified by qRT-PCR. [score:1]
In them, 7 miRNAs (mmu-let-7k, mmu-miR-378d, mmu-miR-129-1-3p, mmu-miR-21a-5p, mmu-miR-29c-3p, mmu-miR-203-3p, and mmu-miR-7a-5p) were identified in both comparison groups (Fig.   2). [score:1]
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44
[+] score: 10
Other miRNAs from this paper: hsa-mir-7-1, hsa-mir-7-2, hsa-mir-7-3, mmu-mir-7a-1, mmu-mir-7b
In zebrafish, which do not have an endogenous copy of ciRS-7 but do express miR-7, ectopic expression of the ciRS-7 sequence results in a defect in midbrain development [6]. [score:6]
The sequence of ciRS-7 is highly repetitive with over 70 repeated miR-7 seed sequences in humans, most of which are conserved across eutherian mammals, and its expression is highly variable across tissues [6– 8]. [score:3]
Indeed, the cytoplasmic ciRS-7 circles have been shown to function by sequestering mir-7 [6, 7], a mechanism unlikely to be employed by nuclear ASINC. [score:1]
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45
[+] score: 9
Among those, were miR-7a, miR-7b, miR-146b, miR-199a and miR-199b* that were regulated at very early time-points after induction of MSCs and show the same direction of up- or down-regulation in the NIH/3T3 mo del. [score:6]
Five of these eight miRNAs (miR-199a, miR-7, miR-146b, miR-7b, and miR-199b*) were similarly regulated early during the adipogenesis process of MSCs, indicating that their regulation was due to cell cycle arrest rather than adipogenesis. [score:3]
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[+] score: 9
Interestingly, miR-7 is a potent tumor suppressor in breast cancer cells [79] consistent with the observed ability of ADAM8 to repress its expression. [score:5]
Out of these 68 miRNAs, two (miR-324 and miR-7) were found to be downregulated by ADAM8. [score:4]
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47
[+] score: 9
Other miRNAs from this paper: hsa-mir-7-1, hsa-mir-7-2, hsa-mir-7-3, mmu-mir-7a-1, mmu-mir-7b
For example, the transcription factor Yan represses the expression of a miRNA, miR-7, whereas miR-7 represses Yan expression. [score:5]
This reciprocal negative feedback loop facilitates exclusive cell fate determination during development, with Yan in progenitor cells and miR-7 in photoreceptor cells. [score:2]
Further studies suggested that miR-7 participates in several interlocking feedback and feed-forward loops to buffer against environmental variation during development [26]. [score:2]
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[+] score: 9
Foxp3 repression of SATB1 is achieved by inducing miRNA (miR-155, miR-21, miR-7, miR-34a, and miR-18a) binding to the Satb1 3′ untranslated region, indirectly suppressing SATB1. [score:6]
Four other miRNA, miR-7, miR-18a, miR-34a, and miR-155, have been shown to contribute to the stabilization of Treg suppressor function (47, 55, 56). [score:3]
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[+] score: 9
Among the miRs reported to regulate α-synuclein, miR-7a-5p did not change and miR-153-3p was upregulated in DA neurons from old mice, however, there was no evidence for a reduction of Snca expression (Supplementary Figure S1A). [score:7]
Several miRs, such as miR-133b, miR-7 and miR-153, miR-433, let-7a-5p and miR-184-5p, miR-205, miR-132 and miR-34b/c, have previously been implicated in the development and maintenance of DA neurons and were linked to neurodegeneration. [score:2]
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Accumulations of miR-497 in the cerebellum, of miR-7 in the hypothalamus, and of miR-221 and miR-222 in the hippocampus have also been described in mice [39] and zebrafish (larval and adult brain), where miR-222 is expressed in specific groups of differentiating cells in the rostral parts of the brain [42]. [score:3]
It has recently been shown that the insulin-like growth factor 1 (IGF-1) is targeted by miR-206 [49] and that miR-7 is a repressor of insulin receptor substrate 1 (IRS1) and 2 (IRS2) [50]. [score:3]
The close interdependence between the actions of IGF-1 and IRS1/IRS2 suggests that miR-206 and miR-7 could be involved in tissue or cell-specific regulation of the functions mediated by IGF-1-signalling pathways in the brain. [score:2]
One example is a potential functional cross-talk between miR-206 (enriched in the cerebellum) and miR-7 (enriched in the hypothalamus and amygdala). [score:1]
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[+] score: 8
miR-148b −2.6183 decrease apoptosis miR-152 −2.3341 Increase cell growth miR-17 −6.2545 Bcl2, N-myc miR-181c −3.5756 proliferation and remo deling of muscles miR-190b −2.2 Binds to Ubiquitin-specific protease 46, increase cell growth miR-192 −2.4871 Increase cell growth miR-199a-3p −1.9 Activin receptor IIA, Map3k4 miR-218-1 −2.2887 Increase cell growth miR-23b −2.1623 Increase Cell growth, proliferation miR-26a −2.4565 decrease proapoptotic signaling miR-27a −2.7 Ubiquitin-conjugating enzyme E2N miR-27b −3 Ubiquitin-conjugating enzyme E2N miR-296-3p −7.3378 Increase cell growth, decrease apoptosis miR-322 8.7 Hydroxysteroid (17-beta) dehydrogenase 7 miR-455 129.249 Up-regulated brown adipocyte differentiation miR-470 3.2 TGFB -induced factor homeobox 1 miR-715 18.25 Fucosyltransferase 1 miR-7a −6.2174 Increase cell growth, decrease apoptosis miR-93 −48.423 Map3k14 (NIK) miR-98 1.8 Tripartite motif-containing 6, insulin-like growth factor 2 mRNA binding protein 1 A) C2C12 myotubes were treated with 10ng/ml of TWEAK for 18h following isolation of total RNA enriched with small RNAs. [score:4]
miR-148b −2.6183 decrease apoptosis miR-152 −2.3341 Increase cell growth miR-17 −6.2545 Bcl2, N-myc miR-181c −3.5756 proliferation and remo deling of muscles miR-190b −2.2 Binds to Ubiquitin-specific protease 46, increase cell growth miR-192 −2.4871 Increase cell growth miR-199a-3p −1.9 Activin receptor IIA, Map3k4 miR-218-1 −2.2887 Increase cell growth miR-23b −2.1623 Increase Cell growth, proliferation miR-26a −2.4565 decrease proapoptotic signaling miR-27a −2.7 Ubiquitin-conjugating enzyme E2N miR-27b −3 Ubiquitin-conjugating enzyme E2N miR-296-3p −7.3378 Increase cell growth, decrease apoptosis miR-322 8.7 Hydroxysteroid (17-beta) dehydrogenase 7 miR-455 129.249 Up-regulated brown adipocyte differentiation miR-470 3.2 TGFB -induced factor homeobox 1 miR-715 18.25 Fucosyltransferase 1 miR-7a −6.2174 Increase cell growth, decrease apoptosis miR-93 −48.423 Map3k14 (NIK) miR-98 1.8 Tripartite motif-containing 6, insulin-like growth factor 2 mRNA binding protein 1 In order to understand the interaction between different genes, we generated common networks using Ingenuity Pathway Analysis (IPA) software. [score:4]
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52
[+] score: 8
Other miRNAs from this paper: mmu-mir-145a, mmu-mir-150, mmu-mir-7a-1, mmu-mir-7b, mmu-mir-145b
For instance, circRNA ciRS-7, containing more than 70 conserved miRNA target sites, can sponge miR-7 and thus depress the expression of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) gene, leading to degrade the protein levels of β-amyloid precursor protein (APP) and β-site APP cleaving enzyme 1 (BACE1), suggesting that ciRS-7 may represent a useful target in the development of therapeutic strategies for AD [6, 10]. [score:8]
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53
[+] score: 8
MicroRNA Function Target/s Reference microRNAs involved in neural development let-7 Promotes neuronal differentiation HMGA, LIN28, TLX Nishino et al. (2008), Rybak et al. (2008), Zhao et al. (2010) Neural tube closure MLIN41 Maller Schulman et al. (2008) miR-7a Inhibits differentiation of forebrain dopaminergic neurons PAX6 de Chevigny et al. (2012) miR-9 Promotes neuronal differentiation FOXG1, TLX, STAT3, REST, FGF8, FGFR1, FOXP2 Krichevsky et al. (2006), Leucht et al. (2008), Packer et al. (2008), Shibata et al. (2008), 2011, Zhao et al. (2009), Yoo et al. (2011), Clovis et al. (2012) Promotes proliferation of early human embryonic stem cell-derived neural progenitor cells STMN1 Delaloy et al. (2010) miR-9* Promotes neuronal differentiation BAF53a Yoo et al. (2009), 2011 ? [score:6]
miR-7a regulation of Pax6 controls spatial origin of forebrain dopaminergic neurons. [score:2]
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54
[+] score: 8
As shown in table 1, 20 microRNAs were significantly deregulated in the knockout mice compared to wild type littermate controls, with three miRNAs differing more than two fold, miR-7 being up-regulated and miR-709 and miR-449b being down-regulated in the antrum of Gastrin knockout mice compared to wild types. [score:8]
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55
[+] score: 8
Other miRNAs from this paper: mmu-mir-7a-1, mmu-mir-7b
To study how GPRC5A suppresses EGFR synthesis, we examined the luciferase activities between wild-type and Gprc5a [−/−] LBE cells after transfecting the reporter vector encoding the 3′-untranslated regions (UTRs) of Egfr at the downstream of the luciferase-coding sequence since previous studies indicated that EGFR translation was regulated on hypoxia through 3′-UTR -mediated HIF-2a–RBM4–eIF4E2 formation 16 23, as well as by miR-7 (ref. [score:8]
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56
[+] score: 8
Li and Carthew reported reciprocal negative regulation between Yan protein and miR-7 in Drosophila retinal cells: Yan directly represses the expression of miR-7 in undifferentiated progenitor cells and miR-7 directly represses the expression of Yan in differentiated photoreceptor cells [7]. [score:8]
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57
[+] score: 7
Other miRNAs from this paper: mmu-mir-7a-1
Studies in rodents have shown that Pax6 is necessary for this specifying phenotype (Dellovade et al., 1998; Kohwi et al., 2005; Brill et al., 2008; Haba et al., 2009), and regulation of Pax6 expression by miR-7a is important for fine-tuning the ability of Pax6 to direct phenotype specification (de Chevigny et al., 2012a). [score:5]
miR-7a regulation of Pax6 controls spatial origin of forebrain dopaminergic neurons. [score:2]
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58
[+] score: 7
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-20a, hsa-mir-23a, hsa-mir-24-1, hsa-mir-24-2, hsa-mir-26a-1, hsa-mir-26b, hsa-mir-29a, hsa-mir-30a, hsa-mir-93, hsa-mir-101-1, hsa-mir-29b-1, hsa-mir-29b-2, hsa-mir-107, hsa-mir-16-2, mmu-let-7g, mmu-let-7i, mmu-mir-15b, mmu-mir-23b, mmu-mir-29b-1, mmu-mir-30a, mmu-mir-30b, mmu-mir-101a, mmu-mir-124-3, mmu-mir-125a, mmu-mir-130a, mmu-mir-9-2, mmu-mir-135a-1, mmu-mir-136, mmu-mir-138-2, mmu-mir-140, mmu-mir-144, mmu-mir-145a, mmu-mir-146a, mmu-mir-149, mmu-mir-152, mmu-mir-10b, mmu-mir-181a-2, mmu-mir-182, mmu-mir-183, mmu-mir-185, mmu-mir-24-1, mmu-mir-191, mmu-mir-193a, mmu-mir-195a, mmu-mir-200b, mmu-mir-204, hsa-mir-30c-2, hsa-mir-30d, mmu-mir-30e, 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-183, hsa-mir-204, hsa-mir-181a-1, hsa-mir-221, hsa-mir-222, hsa-mir-200b, mmu-mir-301a, mmu-mir-34c, mmu-mir-34b, mmu-let-7d, mmu-mir-130b, hsa-let-7g, hsa-let-7i, hsa-mir-15b, hsa-mir-23b, hsa-mir-30b, hsa-mir-124-1, hsa-mir-124-2, hsa-mir-124-3, hsa-mir-130a, hsa-mir-135a-1, hsa-mir-135a-2, hsa-mir-138-2, hsa-mir-140, hsa-mir-144, hsa-mir-145, hsa-mir-152, hsa-mir-191, hsa-mir-9-1, hsa-mir-9-2, hsa-mir-9-3, hsa-mir-125a, hsa-mir-136, hsa-mir-138-1, hsa-mir-146a, hsa-mir-149, hsa-mir-185, hsa-mir-193a, hsa-mir-195, hsa-mir-320a, 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-20a, mmu-mir-23a, mmu-mir-24-2, mmu-mir-26a-1, mmu-mir-26b, mmu-mir-29a, mmu-mir-29c, mmu-mir-93, mmu-mir-34a, mmu-mir-330, mmu-mir-339, mmu-mir-340, mmu-mir-135b, mmu-mir-101b, hsa-mir-200c, hsa-mir-181b-2, mmu-mir-107, mmu-mir-10a, mmu-mir-17, mmu-mir-200c, mmu-mir-181a-1, mmu-mir-320, mmu-mir-26a-2, mmu-mir-221, mmu-mir-222, mmu-mir-29b-2, mmu-mir-135a-2, mmu-mir-124-1, mmu-mir-124-2, mmu-mir-9-1, mmu-mir-9-3, mmu-mir-138-1, mmu-mir-181b-1, mmu-mir-181c, mmu-mir-7a-1, mmu-mir-7b, hsa-mir-29c, hsa-mir-30c-1, hsa-mir-101-2, hsa-mir-34b, hsa-mir-34c, hsa-mir-301a, hsa-mir-130b, hsa-mir-30e, hsa-mir-26a-2, hsa-mir-361, mmu-mir-361, hsa-mir-376a-1, mmu-mir-376a, hsa-mir-340, hsa-mir-330, hsa-mir-135b, hsa-mir-339, hsa-mir-335, mmu-mir-335, mmu-mir-181b-2, mmu-mir-376b, mmu-mir-434, mmu-mir-467a-1, hsa-mir-376b, hsa-mir-485, hsa-mir-146b, hsa-mir-193b, hsa-mir-181d, mmu-mir-485, mmu-mir-541, hsa-mir-376a-2, hsa-mir-320b-1, hsa-mir-320c-1, hsa-mir-320b-2, mmu-mir-301b, mmu-mir-674, mmu-mir-146b, mmu-mir-467b, mmu-mir-669c, mmu-mir-708, mmu-mir-676, mmu-mir-181d, mmu-mir-193b, mmu-mir-467c, mmu-mir-467d, hsa-mir-541, hsa-mir-708, hsa-mir-301b, mmu-mir-467e, mmu-mir-467f, mmu-mir-467g, mmu-mir-467h, hsa-mir-320d-1, hsa-mir-320c-2, hsa-mir-320d-2, mmu-mir-467a-2, mmu-mir-467a-3, mmu-mir-467a-4, mmu-mir-467a-5, mmu-mir-467a-6, mmu-mir-467a-7, mmu-mir-467a-8, mmu-mir-467a-9, mmu-mir-467a-10, hsa-mir-320e, hsa-mir-676, mmu-mir-101c, mmu-mir-195b, mmu-mir-145b, mmu-let-7j, mmu-mir-130c, mmu-mir-30f, mmu-let-7k, mmu-mir-9b-2, mmu-mir-124b, mmu-mir-9b-1, mmu-mir-9b-3
The miRNA families that change expression in both mouse and human were: let-7, miR-7, miR-15, miR-101, miR-140, miR-152 (all validated by qPCR, P < 0.05), as well as miR-17, miR-34, miR-135, miR-144, miR-146, miR-301, miR-339, miR-368 (qPCR not performed). [score:3]
The miRNA families that change expression in both mice and rats were: mir-7, mir-9, mir-10, mir-15, mir-17, mir-26, mir-29, mir-30, mir-101, mir-130, mir-181, mir-204, mir-339, mir-340, mir-368, mir-434, mir-467. [score:3]
88E-0327mmu-miR-451mir-4510.3310.722.79E-046.57E-0366mmu-miR-669c-5pmir-4670.178.847.68E-037.41E-0255mmu-miR-485-3pmir-4850.196.755.52E-036.39E-0265mmu-miR-669nmir-669n0.147.887.46E-037.31E-0260mmu-miR-674-5pmir-6740.178.646.08E-036.45E-0254mmu-miR-676-3pmir-6760.156.634.48E-035.28E-0211mmu-miR-7a-5pmir-70.249.171.37E-057.91E-0436mmu-miR-708-5pmir-7080.179.389.09E-041. [score:1]
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59
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In particular, miR-143 and miR-145 were found to be down-regulated in the colon biopsies of UC patients [41], and miR-7 was observed to be down-regulated in the mucosa of CD patients [42]. [score:7]
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60
[+] score: 7
Other miRNAs from this paper: mmu-mir-7a-1, mmu-mir-7b
On the other hand, Hsia et al found an opposing effect of lapatinib in TNBC that lapatinib may increase the migration and invasion of MDA-MB-231 cells by upregulating EGFR and COX-2 through the downregulation of microRNA-7, providing a potential explanation for the worse clinical outcome of TNBC patients who receive lapatinib -based treatment [62]. [score:7]
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61
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Post-transcriptional regulation of alpha-synuclein expression by mir-7 and mir-153. [score:4]
Moreover, three miRNA (miRNA-7, miRNA-9, and miRNA-106b) were found to be associated with neurodegenerative diseases and only one, namely miRNA-9, with intellectual disability (Doxakis, 2010; Wang et al., 2010; Xu et al., 2011; Hu et al., 2017). [score:3]
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62
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For example, miR-7 suppressed active chronic gastritis and gastric cancer by targeting Cox-2 [26] and miR-1225-5p constrained GC growth and metastatic potential via inhibition of β-catenin signaling [27]. [score:7]
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63
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Target gene Description miRNA ID Osteoblastic genes COL1A1 Type I collagen miR-29a, miR-150, miR-185 BGLAP Osteocalcin – RUNX2 Runt-related transcription factor – Chondrogenic genes COL2A2 Type II collagen miR-7, miR-29a, miR-29b COL10A1 Type X collagen miR-101 SOX9 SRY (sex determining region Y)-box 9 miR-101, miR-124 for the selected genes were carried out with TargetScan, PicTar, or miRanda miRNA target prediction tools. [score:7]
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64
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Herzer et al. identified mir-7a as a hypothalamic-enriched miRNA with a high expression in Neuropeptide Y/Agouti-related orexigenic peptide neurons (Herzer et al., 2012). [score:3]
Locked nucleic acid -based in situ hybridisation reveals miR-7a as a hypothalamus-enriched microRNA with a distinct expression pattern. [score:3]
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65
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On the other hand, miR-7a-5p, miR-15b-5p, miR-105, miR-133b-3p, which were similarly expressed in SAMP8 and SAMR1 mice, were modulated by exercise in both strains (putative markers of exercise unrelated to aging). [score:3]
In addition, we found that miR-7a-5p, miR-15b-5p, miR-105, and miR-133-3p exhibited similar expression levels in sedentary strains but were similarly modulated by exercise in SAMP8 and SAMR1 mice (Figures 2D–G). [score:3]
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66
[+] score: 6
Curcumin inhibits cell growth and invasion through up-regulation of miR-7 in pancreatic cancer cells. [score:6]
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67
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Zhang H et al. found that HOTAIR negatively regulated miR-7 expression and promoted STAT3 and SETDB1 expression, thus promoting the proliferation and invasion of breast cancer stem cells [33]. [score:6]
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68
[+] score: 6
Other miRNAs from this paper: mmu-mir-7a-1
This discrepancy might be due to post-transcriptional or translational regulation, as in a recent report showing that miR-7a can restrict Pax6 protein expression in neural stem cells [34]. [score:6]
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69
[+] score: 5
For instance, ciRS-7 was found to inhibit cancer via miR-7 by down -regulating some critical oncogenes [31]. [score:4]
MiRNA response elements (MREs) were found in some circRNAs, which could sponge miRNAs, such as miR-7, miR-17, and miR-214 [29]. [score:1]
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70
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For example, the circRNA ciRS-7 acts as a sponge for miR-7, and ciRS-7 is resistant to miRNA -mediated target destabilization, thus strongly suppressing miR-7 activity [15]. [score:5]
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71
[+] score: 5
In addition, in glioma cells, miR-7 binds to the EGFR 3’UTR and decreases cell invasiveness by suppressing translation of EGFR [9]. [score:5]
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72
[+] score: 5
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-20a, hsa-mir-21, hsa-mir-29a, hsa-mir-33a, hsa-mir-29b-1, hsa-mir-29b-2, hsa-mir-107, hsa-mir-16-2, mmu-let-7g, mmu-let-7i, mmu-mir-1a-1, mmu-mir-29b-1, mmu-mir-124-3, mmu-mir-126a, mmu-mir-9-2, mmu-mir-132, mmu-mir-133a-1, mmu-mir-134, mmu-mir-138-2, mmu-mir-145a, mmu-mir-152, mmu-mir-10b, mmu-mir-181a-2, hsa-mir-192, mmu-mir-204, mmu-mir-206, hsa-mir-148a, mmu-mir-143, hsa-mir-7-1, hsa-mir-7-2, hsa-mir-7-3, hsa-mir-10b, hsa-mir-34a, hsa-mir-181a-2, hsa-mir-181b-1, hsa-mir-204, hsa-mir-211, hsa-mir-212, hsa-mir-181a-1, mmu-mir-34c, mmu-mir-34b, mmu-let-7d, mmu-mir-106b, hsa-let-7g, hsa-let-7i, hsa-mir-1-2, hsa-mir-124-1, hsa-mir-124-2, hsa-mir-124-3, hsa-mir-132, hsa-mir-133a-1, hsa-mir-133a-2, hsa-mir-138-2, hsa-mir-143, hsa-mir-145, hsa-mir-152, hsa-mir-9-1, hsa-mir-9-2, hsa-mir-9-3, hsa-mir-126, hsa-mir-134, hsa-mir-138-1, hsa-mir-206, mmu-mir-148a, mmu-mir-192, 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-20a, mmu-mir-21a, mmu-mir-29a, mmu-mir-29c, mmu-mir-34a, mmu-mir-330, hsa-mir-1-1, mmu-mir-1a-2, hsa-mir-181b-2, mmu-mir-107, mmu-mir-17, mmu-mir-212, mmu-mir-181a-1, mmu-mir-33, mmu-mir-211, mmu-mir-29b-2, mmu-mir-124-1, mmu-mir-124-2, mmu-mir-9-1, mmu-mir-9-3, mmu-mir-138-1, mmu-mir-181b-1, mmu-mir-7a-1, mmu-mir-7b, hsa-mir-106b, hsa-mir-29c, hsa-mir-34b, hsa-mir-34c, hsa-mir-330, mmu-mir-133a-2, mmu-mir-133b, hsa-mir-133b, mmu-mir-181b-2, hsa-mir-181d, hsa-mir-505, hsa-mir-590, hsa-mir-33b, hsa-mir-454, mmu-mir-505, mmu-mir-181d, mmu-mir-590, mmu-mir-1b, mmu-mir-145b, mmu-mir-21b, mmu-let-7j, mmu-mir-21c, mmu-let-7k, mmu-mir-126b, mmu-mir-9b-2, mmu-mir-124b, mmu-mir-9b-1, mmu-mir-9b-3
MiR-7, which negatively controls the function of α-synuclein mRNA (Mouradian, 2012), also serves as an anti-metastatic miRNA in gastric cancer by targeting Insulin-like Growth Factor-1 (IGF-1) (Zhao et al., 2012). [score:3]
MicroRNA-7 functions as an anti-metastatic microRNA in gastric cancer by targeting insulin-like growth factor-1 receptor. [score:2]
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73
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Li et al. previously demonstrated removal of mir-7 had no observed phenotypic effect in Drosophila sensory cells under standard laboratory conditions [30], just as many miRNA knockouts or knockdowns have no observable phenotype. [score:3]
However when Li et al. perturbed embryos by regularly varying the temperature, miR-7 mutant flies had abnormal sensory cells, demonstrating that the hybrid feed forward/feedback motif containing miR-7 imparted robustness to sensory cell development. [score:2]
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74
[+] score: 5
For example, ciRS-7 contains miRNA-7 binding sites, thereby suppressing miR-7 activity, resulting in increased levels of miR-7 targets [21]. [score:5]
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75
[+] score: 5
As shown in Figure  2, miRNAs known to be expressed at high level in beta-cells [26- 28], such as miR-7, miR-29a and miR-146a, were released in exosomes by MIN6B1 cells and human islets. [score:3]
The amount of miR-7, miR-29a and miR-146a recovered in exosomes was measured by qPCR and is expressed as percentage of the corresponding miRNA present inside MIN6B1 cells (A) or human islets (B). [score:1]
As expected, miR-7, miR-29a and miR-146a released by MIN6B1 cells were protected from confirming that they resided inside the exosomes (Figure  2C). [score:1]
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76
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Fang et al. reported that miR-7 can inhibit HCC tumor growth and metastasis by targeting PIK3CD [46]. [score:5]
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77
[+] score: 5
For instance, miR-273 and lsy-6 regulate the expression of taste receptors in C. elegans chemosensory neurons [7, 8], while Drosophila miR-7 regulates photoreceptor cell differentiation [9]. [score:5]
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In addition, miR-7, miR-153 and miR-155 negatively regulated α-syn expression, which is a crucial regulator for neuroinflammation in PD [15, 24]. [score:5]
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79
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Sequence-specific stimulation of TNFa secretion for mir-7a and 29b was confirmed in RAW264.7 mouse macrophages in vitro (Fig. 1D). [score:1]
The presence of beta cell miRNAs i. e. miR-375, miR-29b, and miR-7a in MIN6 exosomes was confirmed by RT-qPCR (S6C in File S1). [score:1]
Interestingly, secretion of the anti-inflammatory cytokine IL-10 was also observed for miR-29b and miR-7a (Fig. 1C). [score:1]
Three miRNA sequences, namely miR-29b, miR-7a, and miR-376a induced IL-12 secretion (Fig. 1A) and enhanced basal TNFa secretion (Fig. 1B), exceeding levels obtained for LPS and siRNA9.2 [21] positive controls. [score:1]
Again, miR-29b, miR-7a and miR-376a stimulated IFNa production in sera of treated mice, in contrast to miR-127 and miR-210. [score:1]
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80
[+] score: 4
These searches identified 8 miRNAs (miR-7a, miR-7b, miR-28, miR-186, miR-381, miR-876, miR-543, and miR-708) that might target CRX. [score:3]
control miR-7b mimcs CRX 1.005 ± 0.090.43 ± 0.04 [*] miR-7b 1.00 ± 0.042.62 ± 0.16 [*] Con miR-186 mimics CRX 1.05 ± 0.14 1.07 ± 0.09 miR-186 1.00 ± 0.021.25 ± 0.02 [*] Con miR-7a mimics CRX 1.02 ± 0.08 0.91 ± 0.016 miR-7a 1.01 ± 0.15 1.15 ± 0.06 Con miR-876 mimics CRX 1.00 ± 0.04 1.14 ± 0.05 miR-876 1.00 ± 0.09 1.25 ± 0. 14 Con miR-708 mimics CRX 1.00 ± 0.11 1.02 ± 0.10 miR-708 1.00 ± 0.03 0.89 ± 0.07 Con miR-381 mimics CRX 1.01 ± 0.10 1.25 ± 0.15 miR-381 1.00 ± 0.01 0.83 ± 0.09 Con miR-543 mimics CRX 1.00 ± 0.060.39 ± 0.02 [*] miR-543 1.09 ± 0.142.56 ± 0.18 [*] Con miR-28 mimics CRX 1.00 ± 0.040.36 ± 0.02 [*] miR-28 1.00 ± 0.092.08 ± 0. 10 [*] * P < 0.05. [score:1]
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81
[+] score: 4
Moreover, some differentially up-regulated microRNAs (such as miR-466h-5p, miR-135a-1*, miR-2137, miR-223, miR-139-5p, miR-29b-1*, and miR-7a) displayed earlier in PR8 infected lungs than in BJ501 infected lungs. [score:4]
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82
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For example, Zhou et al. revealed that miR-7 regulated neuroinflammation in the pathogenesis of PD by targeting Nod-like receptor protein 3 inflammasome [16]. [score:4]
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83
[+] score: 4
The other 5 miRNAs all showed significant alterations in expression levels at 9 months of age, but these alterations became either not significant (miR-331-3p, miR-7a-5p, miR-501-3p and miR-434-3p) or reversed (miR-7b-5p) at 12 months of age. [score:3]
Among them, nine miRNAs (miR-99b-5p, miR-7b-5p, miR-7a-5p, miR-501-3p, miR-434-3p, miR-409-5p, miR-331-3p, miR-138-5p and miR-100-5p) showed consistent changes in both groups. [score:1]
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84
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For example miR-7a showed significant association with two loci present on chromosome 15 (3–9 Mb, CEL. [score:1]
The highest -log P value of 10.38 was found between the SNP pairs rs13480360 (chr 10, ~67 Mb, nearest gene: AK139516) and rs3689658 (chr 2, ~ 85 Mb, nearest gene: Olfr1006) for miR-7a (Table  2). [score:1]
In total, we found 119 SNP pairs for miRNA miR-7a. [score:1]
The hub locus (i. e. SNP with the maximal number of interactions) for miR-7a was observed on chromosome 16 (rs3680665 ~ 84 Mb, nearest gene: AK04263). [score:1]
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85
[+] score: 4
Other miRNAs from this paper: mmu-mir-92a-2, mmu-mir-92a-1, mmu-mir-7a-1, mmu-mir-7b, mmu-mir-92b
Regulation of human PAX6 expression by miR-7. Evol Bioinform Online. [score:4]
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86
[+] score: 4
Post-transcriptional regulation of alpha-synuclein expression by mir-7 and mir-153. [score:4]
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87
[+] score: 4
Our data revealed that expression levels of miR7 and miR125b were unchanged in ED16, 4W and 14W lenses. [score:3]
In previous research [27, 29, 30], several miRNAs such as miR124, miR7, miR125b and let7b have been detected in rat lens and in regeneration of new lens by transdifferentiation of pigment epithelial cells of the dorsal iris. [score:1]
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88
[+] score: 4
Other miRNAs from this paper: mmu-mir-7a-1, mmu-mir-7b
MicroRNA-7 targets Nod-like receptor protein 3 inflammasome to modulate neuroinflammation in the pathogenesis of Parkinson’s disease. [score:4]
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89
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miRNAs involved in embryonic and adult neurogenesis such as mir-137, mir-128, mir-124a, mir-326, or mir-7 were found significantly downregulated by VPA. [score:4]
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90
[+] score: 4
Other miRNAs from this paper: hsa-mir-7-1, hsa-mir-7-2, hsa-mir-7-3, mmu-mir-7a-1, mmu-mir-7b
Suggestive of a regulatory role for the mouse variants is developmental stage changes in their distribution in heart and kidney, and localization of the 1b variant specifically to the hippocampus where its 5′ end is a target of the micro RNA Mir7 [20]. [score:4]
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91
[+] score: 3
For example, CDR1as participates in colorectal cancer and hepatocellular carcinoma by sequestering miR-7 away from its targets [27, 28]. [score:3]
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92
[+] score: 3
CDR1 antisense transcript (CDR1as) is a circular RNA in mouse and human brain that contains more than 70 binding sites for the microRNA miR-7 and may suppress its activity. [score:3]
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93
[+] score: 3
As a control, the total list of miRNAs profiled was randomized in order and 9 miRNAs were selected (miR-452, miR-7, miR-205, miR-15a, miR-144, miR-183, miR-463, miR-25, miR-99a), targets and pathway ontology was analyzed as for the candidate list. [score:3]
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94
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26 were not detected in the knockout including the most abundant islet miRNAs, miR-375 and miR-7a. [score:2]
These include the islet-specific miR-375 and another abundant islet miRNA, miR-7a [28]. [score:1]
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95
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Recently, a substantial number of deregulated miRNAs including miR-106b-25 cluster, miR-21, miR-218, miR-7, and miR-335 have been identified as modulators of cell growth, apoptosis, migration, or invasion in gastric cancer development [11]– [15]. [score:3]
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96
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miRNA Fold change at 3 dpi Fold change at 5 dpi mmu-miR-466h-3p NS (Not significant) 14.311053 mmu-miR-346-5p NS 3.4766614 mmu-miR-877-3p NS 3.416667 mmu-miR-7a-5p NS 2.1413074 mmu-miR-5107-5p NS −2.047792 mmu-miR-3473a −2.2872427 −2.1317267 mmu-miR-150-5p NS −2.1770155 mmu-miR-3473b −3.2475147 −2.282881 mmu-miR-721 NS −2.6864858 mmu-miR-669b-5p NS −2.9408455 mmu-miR-709 NS −3.0065749 mmu-miR-669n NS −3.0094464 mmu-miR-468-3p NS −3.40051 mmu-miR-466m-5p NS −4.33538 mmu-miR-32-3p NS −4.5324426 mmu-miR-466h-5p NS −4.9673104 mmu-miR-3082-5p NS −6.01648 mmu-miR-466i-5p NS −7.6776285 mmu-miR-1187 NS −8.772696 mmu-miR-574-5p NS −9.259378 To confirm the validity of the differentially expressed miRNAs that had been identified by microarray analysis, we performed real-time PCR on all 20 of these miRNAs using the polyA tailing technique. [score:3]
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97
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MiR-7 has been reported to target α-synuclein mRNA and control the level of α-synuclein protein, leading to the increased resistance to oxidative stress [44]. [score:2]
This may involve specific individual miRNAs, such as miR-133b, miR-7, miR-184-5p, miR-153, and others, which are implicated in maintaining DA neuronal homeostasis and involved in the pathogenesis of PD 40– 42. [score:1]
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98
[+] score: 3
Other miRNAs from this paper: mmu-mir-34a, mmu-mir-7a-1, mmu-mir-7b
miR-7 suppresses brain metastasis of breast cancer stem-like cells by modulating KLF4. [score:3]
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99
[+] score: 3
In relation to the eye, miR-7 has been shown to play an important role in photoreceptor differentiation in Drosophila [25] and other miRs, such as miR-9, miR-96, miR-124a, miR-181, miR-182, and miR-183, were found to be highly expressed during morphogenesis of the zebrafish eye [16]. [score:3]
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100
[+] score: 3
miR-7 reverses epithelial–mesenchymal transition by inactivating AKT/ERK1/2 via EGFR and inhibits OVCa metastasis [43]. [score:3]
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