6 publications mentioning mmu-mir-3473a

Open access articles that are associated with the species Mus musculus and mention the gene name mir-3473a. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1

As Fig.   1d and Fig.   5b shown, Bt can upregulate TNF-α mRNA expression at the late infectious phase (>16 hpi), but it would not change TNF-α expression after treatment of miR-3473 mimics or inhibitors like that in Bp -treated macrophages (Fig.   5b).

e qRT-PCR analysis of miR-3473 expression in macrophages transfected with mimic, inhibitor, mimic control or inhibitor control oligonucleotides of miR-3473.

This way, we hypothesized that miR-3473 regulated TNF-α expression through targeting TRAF3 rather than direct anchoring.

In addition, TargetScan suggested that miR-3473 possibly target TRAF3 (TNF receptor -associated factor 3), a well-known negative regulator of the NF-κB pathway, which was probably involved in the TNF-α induction and apoptosis in cells with Bp infection.

Since TRAF3 has been known as a negative regulator of NF-κB pathway, the mechanism for how miR-3473 regulates TNF-α expression is unknown.

siRNAs or oligonucleotides (miR-3473 mimics, inhibitors, mimic control and inhibitor control) were purchased from Ribobio Corporation (Guangzhou, China) and suspected oligonucleotide was delivered through aerosol inhalation (15 μg in 50 μL PBS/every dose) [22].

a qRT-PCR analysis of TNF-α mRNA expression in Bp-infected or uninfected macrophages pre -treated with miR-3473 mimic, inhibitor or controls.

miR-3473 mimic and inhibitor were used to change the expression level of miR-3473.

While, among the list of targets of miR-3473, TNF receptor -associated factor 3 (TRAF3) was included and suggested it should be a potential target of miR-3473 (Fig.   3a).

This indicated that miR-3473, as an independent regulation factor, can play a key role in Bp -induced TNF-α expression and cell apoptosis.

These results suggested that miR-3473 manipulated TRAF3-NF-κB-TNF-α regulation axis to affect TNF-α expression in Bp-infected macrophages.

In addition, miR-3473 inhibitor was administered into mice and its regulation on TNF-α release was equally obvious like that in vitro (Fig.   6c).

TRAF3, not TNF-α, was a direct target of miR-3473.

After luciferase assay on macrophages which were carrying the 3ˈ-UTR TRAF3 luciferase reporter, it was found that induction of miR-3473 would significantly inhibit the luciferase activities but there was no inhibition in those macrophages transfected with the mutant 3ˈ-UTR TRAF3 luciferase reporter (Fig.   3f).

a Protein levels of TRAF3, Phospho-NF-κB p65 and NF-κB p65 in Bp-infected or uninfected macrophages transfected with miR-3473 mimic, inhibitor or controls, as assessed by western blot with Actin as an internal control.

A mimic and inhibitor of miR-3473 were transfected into macrophages using Lipofectamine 2000™, and transfection efficiency was confirmed by qRT-PCR (Fig.   3e).

In vivo, it was found that miR-3473 expression of total lungs cells from Bp -treated was higher than that from Bt -treated mice.

To verify that TRAF3 is a molecular target of miR-3473, a fragment of the 3ˈ-UTR of TRAF3, containing the putative miR-3473 binding site (Fig.   3a) or a mutant miR-3473 binding site (Fig.   3c), was individually cloned into a pMIR-Report luciferase plasmid, with pRL-TK as an internal control.

a qRT -PCR analysis of miR-3473 expression in Bp, Bt-infected or uninfected macrophages at different time post infection (8 h, 12 h, 24 h, MOI = 10).

d Survival curves of mice infected with Bp, Bt or PBS for 7 days with or without treatment of miR-3473 inhibitor oligonucleotides.

And miR-3473 inhibitor was able to decrease the TNF-α release of mice and prolong the survival of mice with Bp infection.

Burkholderia pseudomallei (Bp) Burkholderia thailandensis (Bt) TNF-α Apoptosis miR-3473 TRAF3 NF-κB Burkholderia pseudomallei (Bp) is the causative agent of the human disease melioidosis, which is endemic in southeast Asia and northern Australia, with manifestations ranging from fever to pneumonia to life-threatening sepsis.

a The putative binding sites of miR-3473 in the TRAF3 3′-UTR are shown (TargetScanMouse 6.2, http://www.

We also tested the expression of miR-3473 in TC-1 cell (a type of murine respiratory epithelial cells) treated by Bp, but it did not changed significantly as that in macrophages (Fig.   2d).

We found that treatment with miR-3473 mimics markedly enhanced TNF-α mRNA expression in uninfected or Bp-infected macrophages (Fig.   5a).

d miR-3473 expression in murine macrophages (RAW264.7 cell line) or epithelial cells (TC-1 cell line) at 12 h post Bp, Bt or no infection (MOI = 10 or 0).

c miR-3473 expression of macrophages treated with different pathogens (MOI = 10) for 12 h post infection, including Salmonella typhi, Escherichia coli DH5a, Bt or 3 strains of clinical Bp (BPC006, BPC011, BPC056, all sequenced by MLST and deposited in http://bpseudomallei.

To confirm the validity of the microarray, the expression levels of TNF-α, TRAF3 and miR-3473 were detected by qRT-PCR.

However, miR-3473 inhibitors had no obvious effect on the intracellular growth of Bp or Bt (Fig.   5d).

It suggested that TRAF3 (3ˈ-UTR) should be a probable target of miR-3473.

As described in material and method, qRT-PCR was applied to test the expression of miR-3473 in murine lung cells.

As described above, miR-3473 expressed significantly and specifically in total lung cells of mice infected with Bp.

b miR-3473 expression in Bp, Bt-infected or uninfected macrophages at 12 h post infection with different dose of Bp or Bt (MOI = 1, 10, 100, 10000).

c Early apoptosis cells were gated through flow cytometry test in macrophages pre -transfected with miR-3473 inhibitor or control at 12 h after Bp or Bt infection.

Conversely, miR-3473 inhibitor oligonucleotides would decrease TNF-α mRNA in Bp-infected macrophages and abrogate the difference of TNF-α mRNA levels between Bp and Bt-infected cells (Fig.   5a).

On the contrary, after intravenous administration with miR-3473 inhibitor, the survival period of Bp-infected mice extended although the death rate could not be altered at the end.

On the contrary, miR-3473 inhibitor would decrease the activity of NF-κB in Bp-infected macrophages, but not in Bt-infected macrophages (Fig.   4a& b).

Based on the above results, we wondered whether miR-3473 alone can influence TNF-α mRNA expression, cell apoptosis and bacterial replication in macrophages.

It suggested that there should be a miR-3473-TRAF3-NF-κB regulation axis which would play a vital role in Bp -mediated inflammatory response in macrophages.

In sum, miR-3473 plays an important role in the differential pathogenicity of Bp and Bt via miR-3473- TRAF3-TNF-α network, and regulates TNF-α release, cell apoptosis and animal survival after Bp treatment.

The fragments of the TRAF3 3ˈ-UTR containing the miR-3473 target site were amplified from genomic DNA (primers details in Table  1) and cloned into the pMIR-Report plasmid downstream of a reporter synthetic Renilla luciferase gene (hRluc) using SpeI and HindIII.

In vivo, the miR-3473- TRAF3-TNF-α network was also regulating the TNF-α release and the survival of mice.

To generate plasmids with one or more mutations in the binding site for miR-3473, the seed regions were mutated from TCTCTCCA to TGTTTGCA at 1186–1193 of the 3ˈ-UTR and synthesized by Sangon (Shanghai, China) followed by cloning into the pMIR-Report plasmid.

It was the same case for the regulation of miR-3473 on cell apoptosis in Bp or Bt-infected macrophages (Fig.   5c).

miR-3473 was involved in TRAF3-NF-κB-TNF-α regulation axis.

and miR-3473 expression was measured by qRT-PCR described as above.

miR-3473 was specifically induced in Bp-infected murine macrophages.

Furthermore, miR-3473 had a detrimental impact on the survival period of mice infected with lethal dose of Bp (Fig.   6d).

We found that miR-3473 was induced higher in lung cells from Bp-infected mice than that from Bt-infected or uninfected mice (Fig.   6b).

These results suggested miR-3473 was indulgent for excessive inflammatory response and associated with acute death of mice after lethal Bp infection.

But, miR-3473 specifically increased in Bp-infected murine macrophages, rather than that in Bt -treated macrophages and increased significantly at 12 hpi (Fig.   2a).

Moreover, other Gram -negative bacterium, like Salmonella typhi and Escherichia coli DH5α, were not able to induce miR-3473 significantly (Fig.   2c).

Analysis of miR-3473 was conducted as described above.

c miR-3473 inhibitor oligonucleotide was administrated to mice (i. n., through breathing) on the day before inoculation and TNF-α release was measured at 4 dpi.

This indicated that miR-3473 was induced specifically in murine macrophages rather than murine pulmonary cell after Bp infection.

Fig. 4Bp infection specifically reduces TRAF3 by miR3473 which activates NF-κB-TNF-α pathway.

It suggested that miR-3473 can manipulate bacteria -induced host inflammatory but do not influence the recycle of intracellular Bp or Bt.

After transfection of miR-3473 mimics, NF-κB pathway was enhanced significantly (showed by phospho-NF-κB p65 level) with or without Bp infection (Fig.   4a).

miR-3473 was induced significantly in lung cells from Bp-infected mice but would not affect murine survival.

In addition, miR-3473 increased in a dose -dependent manner after Bp infection, but no alteration of miR-3473 in Bt-infected cells was observed, even at a high MOI (Fig.   2b).

miR-3473 was responsible for the differences of TNF-α release and cell apoptosis between Bp and Bt.

In addition, we found that miR-3473 was able to affect the TNF-α release, cell apoptosis and inflammatory response via a miR-3473- TRAF3-TNF-α network.

d The putative binding sites of miR-3473 in the TRAF3 3′-UTR were mutated as shown.

d Intracellular bacteria loads of Bp or Bt-infected macrophages transfected with miR-3473 oligonucleotides were counted at different infectious phase (4 h, 8 h, 12 h, 20 h and 28 h).

Among them, miR-3473 was found to be specific to Bp infection and significantly induced along with the infection process both in murine macrophages and pulmonary cell line (TC-1).

miR-3473 was one of them specifically induced, but not significantly changed in Bt -treated macrophages.

2

At day 5 post-infection, there were four up-regulated and 16 down regulated miRNAs respectively, as shown in Table 1. Importantly, the two miRNAs, namely miRNA-3473a and miRNA-3473b, which were down-regulated at 3 days post-infection, were also down-regulated at 5 days post-infection to almost the same extent.

At day 3 post-infection, there were only two down-regulated miRNAs, miRNA-3473a and miRNA-3473b, and no up-regulated miRNAs.

Therefore, the down-regulation of miR-3473a would lead to over -expression of FAK and MMP9, resulting in BBB breaking.

An interesting finding in our study was that there were only two miRNAs identified at 3 dpi, miR-3473a and -3473b, and they were both down-regulated.

Considering that retrograde axonal transport is a major transmission route of EV71 (Chen et al., 2007), the down-regulation of miR-3473a and -3473b at an early stage of EV71 infection could invoke these neural pathways, thereby mediating EV71 transmission into the mouse brain.

In addition, miR-3473a could also regulate focal adhesion and leukocyte trans-endothelial migration by targeting focal adhesion kinase (FAK/PTK2) and matrix metalloproteinase 9 (MMP9) genes respectively (Supporting File 3).

MiR-709 could regulate the chemokine signaling pathway and miR-3473a might regulate leukocyte trans-endothelial migration.

Thus, the opposite expression trend of miRNA-3473a, -3473b, and 709 strongly suggest that these miRNAs do have function on CNS inflammation.

This report identified a number of miRNAs, including those identified in this study, namely miRNA-3473a, -3473b, and 709, which had the exact opposite expression profiles (Gao et al., 2016).

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.

These results indicated that miR-3473a might play important roles in immunologic injury in the CNS upon EV71 infection.

In addition, miR-3473a and 3473b could involve in axon guidance and the Wnt signaling pathway.

3

To validate the up- and downregulated miRNAs identified in the Solexa sequencing, 6 upregulated (mmu-miR-146a-5p, mmu-miR-341-3p, mmu-miR-879-5p, mmu-miR-3470a, mmu-miR-3473a and mmu-miR-3473b) and six downregulated miRNAs (mmu-miR-96-5p, mmu-miR-141-3p, mmu-miR-182-5p, mmu-miR-200a-3p, mmu-miR-200b-3p and mmu-miR-200b-5p), as well as two novel miRNAs (novel-mir-2 and novel-mir-20), were selected.

[29] Further understanding of the contribution of the high brain level of mmu-miR-3473a during prion infection to flotillin -mediated PrP [C] endocytosis will help us further our knowledge of the physical biology and pathology of prion disease.

A search for the possible targets of mmu-miR-3473a in MIRDB (http://www.

In addition to mmu-miR-3473b, other two family numbers, mmu-miR-3473a and mmu-miR-3473e, are also greatly increased in the brains of the three scrapie-infected mouse mo dels, with average fold-change values of 5.3 and 12.3, respectively.

4

Furthermore, the pathway analysis links a group of miRNAs that were differentially expressed in cbs [+/–] retina to oxidative stress pathway such as miR-205, miR-206, miR-217, miR-30, miR-27, miR-214 and miR-3473.

Hcy also induces alteration of miRNAs related to tight junctions signaling such as miR-128, miR-132, miR-133, miR-195, miR-3473, miR-19, miR-200, miR-205, miR-214, miR-217, miR-23, miR-26, miR-29, miR-30, miR-31 AND miR-690.

5

miRNA p-value Fold-Change miR-146a 1.15E-09 −4.82 miR-31 9.66E-08 −4.95 miR-155 1.97E-07 −3.82 miR-2134 2.13E-07 −2.05 miR-711 1.63E-06 −4.10 miR-3473 1.95E-06 −5.62 miR-574-3p 3.32E-06 −2.55 miR-1195 6.59E-06 2.26 miR-27a* 6.89E-06 14.92 miR-27b* 7.35E-06 2.92 miR-34c 1.80E-05 −3.43 miR-1931 3.34E-05 −2.30 miR-874 4.07E-05 −2.01 miR-196b 7.99E-05 2.59 miR-181a-1* 8.02E-05 4.15 miR-187 8.11E-05 2. 02List of miRs whose expression is altered, identified from microarray analysis by comparison of levels in TM [+] and TM [−] DCs which change >2 fold with p<0.0001.

6

miRNAs that showed high variance between samples in the microarray show strong consistency between microarray and qPCR (mmu-miR-208b-3p, mmu-miR-3473a, mmu-miR-709).