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miR-146a-5p Regulated Cell Proliferation and Apoptosis by Targeting SMAD3 and SMAD4.
Protein & Peptide Letters ( IF 1.0 ) Pub Date : 2020-05-01 , DOI: 10.2174/0929866526666190911142926 Meiyu Qiu 1, 2 , Tao Li 1, 3 , Binhu Wang 1 , Hongbin Gong 1 , Tao Huang 1
中文翻译：
miR-146a-5p通过靶向SMAD3和SMAD4来调节细胞的增殖和凋亡。
更新日期：2020-05-01
Protein & Peptide Letters ( IF 1.0 ) Pub Date : 2020-05-01 , DOI: 10.2174/0929866526666190911142926 Meiyu Qiu 1, 2 , Tao Li 1, 3 , Binhu Wang 1 , Hongbin Gong 1 , Tao Huang 1
Affiliation
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Background: microRNAs (miRNAs) are a small, endogenous non-coding RNAs that are involved in post-transcriptional gene regulation of many biological processes, including embryo implantation and placental development. In our previous study, miR-146a-5p was found expressed higher in the serum exosomes of pregnant sows than non-pregnant. The research on miR-146a-5p has been mainly related to human diseases, but there are few studies on its effects on the reproduction of sows in early pregnancy.
Objective: In this article, our motivation is to study the role of miR-146a-5p in the early pregnancy of sows on the cell proliferetion and apoptosis by targeting SMAD3 and SMAD4. Methods: Bioinformatics software was used to identify the target genes of miR-146a-5p. The wildtype and mutant-type recombinant plasmids of dual-luciferase reporter with 3'-UTR of Smad3 or 3'- UTR of Smad4 were constructed, and co-transfected in porcine kidney cell (PK-15 cell) with miR- 146a-5p mimic, mimic-NC(M-NC), inhibitor and inhibitor-NC(IN-NC), then dual-luciferase activity analysis, qRT-PCR and Western blot were performed to verify the target genes. After the transfection of BeWo choriocarcinoma cell (BeWo cell) with miR-146a-5p mimic, M-NC, inhibitor and IN-NC, the mRNA expression of Caspase-3, BAX and Bcl-2 was measured using qRT-PCR, and the cell proliferation was measured using CCK-8 kit. Results: The luciferase, mRNA and protein expression of Smad3 in PK-15 cells treated by Smad3- 3'-UTR-W co-transfected with miR-146a-5p mimic were significantly lower than that with miR- 146a-5p M-NC, and the results of Smad4 were similar to Smad3, but the protein expression had a trend to lower in mimic group. The expression level of Bcl-2 in the miR-146a-5p mimic group was significantly lower than that in the miR-146a-5p M-NC group, but the expression pattern of Caspase-3 was just opposite. The mimic of miR-146a-5p reduced the proliferation of BeWo cells, however the inhibitor increased. Conclusion: Smad3 and Smad4 are the direct target genes of miR-146a-5p. The expression of Smad3 and Smad4 were affected by the mimic and inhibitor of miR-146a-5p. miR-146a-5p affects cell apoptosis and proliferation by regulating their target genes. This study provided new data to understand the regulation mechanism of early pregnancy in sows.中文翻译：
miR-146a-5p通过靶向SMAD3和SMAD4来调节细胞的增殖和凋亡。
背景：microRNA（miRNA）是一种小的内源非编码RNA，与许多生物过程（包括胚胎植入和胎盘发育）的转录后基因调控有关。在我们先前的研究中，发现怀孕母猪的血清外泌体中的miR-146a-5p的表达高于未怀孕的母猪。关于miR-146a-5p的研究主要涉及人类疾病，但关于其对妊娠早期母猪繁殖的影响的研究很少。
目的：在本文中，我们的动机是通过靶向SMAD3和SMAD4来研究miR-146a-5p在母猪早期妊娠中对细胞增殖和凋亡的作用。 方法：使用生物信息学软件鉴定miR-146a-5p的靶基因。构建了具有Smad3的3'-UTR或Smad4的3'-UTR的双荧光素酶报告基因的野生型和突变型重组质粒，并与miR- 146a-5p在猪肾细胞（PK-15细胞）中共转染。模拟，模拟NC（M-NC），抑制剂和抑制剂NC（IN-NC），然后进行双荧光素酶活性分析，qRT-PCR和Western blot验证靶基因。用miR-146a-5p模拟物，M-NC，抑制剂和IN-NC转染BeWo绒毛膜癌细胞（BeWo细胞）后，使用qRT-PCR测量Caspase-3，BAX和Bcl-2的mRNA表达，并使用CCK-8试剂盒测量细胞增殖。结果：与miR-146a-5p M-NC共转染的Smad3- 3'-UTR-W共转染的PK-15细胞中，Smad3的荧光素酶，mmad和蛋白表达明显低于miR-146a-5p M-NC。 ，并且Smad4的结果与Smad3相似，但是在模拟组中蛋白质表达有降低的趋势。miR-146a-5p模拟组中Bcl-2的表达水平明显低于miR-146a-5p M-NC组，但Caspase-3的表达模式恰好相反。miR-146a-5p的模拟物减少了BeWo细胞的增殖，但抑制剂增加了。结论：Smad3和Smad4是miR-146a-5p的直接靶基因。Smad3和Smad4的表达受miR-146a-5p的模拟物和抑制剂的影响。miR-146a-5p通过调节靶基因来影响细胞凋亡和增殖。该研究为了解母猪早期妊娠的调控机制提供了新的数据。
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