Title:miR-146a-5p Regulated Cell Proliferation and Apoptosis by Targeting SMAD3 and SMAD4
Volume: 27
Issue: 5
Author(s): Meiyu Qiu, Tao Li, Binhu Wang, Hongbin Gong and Tao Huang*
Affiliation:
- College of Animal Science and Technology, Shihezi University, Shihezi 832000,China
Keywords:
Smad3, Smad4, miR-146a-5p, cell proliferation, apoptosis, luciferase.
Abstract: 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.
