The Regulation Of MIR-26B And MIR-34A Targeting HAS2and Inhbb On Porcine Ovarian Granulosa Cell Apoptosis

Follicular atresia is a very common phenomenon in the mammalian ovary, during follicle development, most follicles (75%-99%) degradation, only a very small number of follicles to mature and ovulate. With the exploration of the research on apoptosis, we gradually understand that granulosa cell apoptosis is the main cause of follicular atresia. In recent years, it has been found that many of the factors that inhibit or promote follicular atresia; the molecular mechanism of regulation of follicular atresia, however, has not been fully elucidated, the future remains to be done further research. Study of follicular development and atresia in humans, for the treatment of infertility, improve has some guiding significance for pregnancy rate; also has a very important significance for improving animal reproduction and embryonic development.Along with the microRNA (miRNA) researched, it was found that miRNAs are a large class of non-coding, an important role in the regulation of functional RNA family. MiRNA is widely distributed in various eukaryotic organisms, involved in cell proliferation, differentiation, apoptosis, embryonic development, metabolism, and tumor inhibition and cell signal transduction and other life activities. It has been confirmed that some miRNA expression is significantly up-regulated and promote apoptosis of porcine ovarian granulosa cells in porcine follicular atresia. Therefore, this article will research the relationship of miRNA and granulosa cell apoptosis, to elucidate the molecular mechanism of regulation of follicular atresia.The study of porcine granulosa cells were selected as the main research object. MiR-26b and miR-34a were selected as control factors of follicular atresia and granulosa cells apoptosis based on both our previous research results and abroad references. We selected HAS2and INHBB as the candidate target genes, by the bioinformatics web predicting target genes and the target gene function. In HeLa229cells, we verified the main effect target gene of miRNA by dual luciferase reporter system and site-directed mutagenesis experiments. Then, with transfected porcine granulosa cells, the mechanism of miR-26b and miR-34a regulating their target genes were verified by the Real-time PCR, flow cytometry and Western Blotting, further to clarify porcine follicular atresia and the apoptosis of granulosa cells regulation. The main results achieved were as follows:1.The expression of target gene HAS2was showed by RT-PCR in porcine ovaries and granulosa cells; It was verified that the HAS2gene was a direct target gene of miR-26b by dual luciferase reporter system and site-directed mutagenesis experiments; The apoptosis was found in granulosa cells transfected with miR-26b by Real-time PCR; The phenomenon was found that miR-26b promoted granulosa cells apoptosis, and miR-26b inhibitor can inhibit endogenous miR-26b on granulosa cell apoptosis. It was confirmed that miR-26b regulates HAS2gene expression at the post-transcriptional level by Real-time PCR and Western Blotting.2.HAS2mRNA expression levels were significantly increased with cumulus expansion during IVM, suggesting its important roles in cumulus expansion and oocyte maturation.3.The expression of target gene INHBB was showed by RT-PCR in porcine ovaries and granulosa cells:It was verified that the INHBB gene was a direct target gene of miR-26b by dual luciferase reporter system and site-directed mutagenesis experiments; It was confirmed that miR-26b regulates HAS2gene expression at the post-transcriptional level by Real-time PCR and Western Blotting.4.It was verified that the INHBB gene was a direct target gene of miR-34a by dual luciferase reporter system and site-directed mutagenesis experiments; The apoptosis was found in granulosa cells transfected with miR-34a by Real-time PCR; The phenomenon was found that miR-34a promoted granulosa cells apoptosis, and miR-34a inhibitor can inhibit endogenous miR-34a on granulosa cell apoptosis. It was confirmed that miR-34a regulates INHBB gene expression at the post-transcriptional level by Real-time PCR and Western Blotting.This paper confirmed that the HAS2and INHBB gene were target genes of miR-26b, also INHBB gene was a target gene of miR-34a.The regulation capability of miR-34a for target gene INHBB was more powerful than miR-26b.MiR-26b and miR-34a can both promote granulosa cells apoptosis, inhibit target genes expression at the post-transcriptional level. The study is helpful to explore the molecular mechanisms of miRNA promote pig granulosa cell apoptosis. In the meanwhile, to expands miRNA function, and contributes to in-depth study of follicular atresia molecular regulation mechanism.