The Regulation Of MiRNA-126-3p On Porcine Ovarian Granulosa Cells Function

The ovaries are important for mammalian gonads, and ovaries are follicle development and ovulation vital organs. Follicles are unit of structure and function of ovary, primordial follicles recruitment, activation and normal growth of follicle development, ovulation is the premise of guarantee the sow lifetime fecundity. Follicle development process along with granulosa cell growth, development and differentiation, proliferation and apoptosis of granulosa cells is closely related to the growth and development of the oocyte, primordial follicles start growing, growing follicular development and follicular atresia. Many studies show that granule cells related genes and miRNA affect granulosa cell function, thereby affecting follicle growth and development?Our previous pre-puberty and suspected by premature ovarian failure(Premature ovarian failure, POF) gilts ovaries were miRNA expression analysis obtained with follicular development-related miRNAs, screened in suspected POF gilts ovaries high expression of miR-126-3p candidate miRNA. In this study, the miR-126-3p and target gene biological function of granulosa cells were investigated by Annexin V-FITC flow cytometry, Edu fluorescence microscopy, q RT-PCR and Western Blot. The results were helpful for better understand the mechanism of miRNA regulation on primordial follicular development. The main results and conclusions are as follows:1. Annexin V-FITC flow cytometry results showed that, miR-126-3p can inhibit apoptosis in granulosa cell. Using the Edu fluorescence microscopy test results show that, miR-126-3p can promote granulosa cell proliferation.2. Bioinformatics prediction targeting miR-126-3p PIK3R2 and TSC1 gene, which is dormant primordial follicle activation and related PI3K-AKT pathway key genes. Further through the Dual-luciferase reporter assay showed that miR-126-3p could bind to the 3' untranslated regions(3'UTR) of PIK3R2 and TSC1 gene; q RT-PCR and western blotting further identified that PIK3R2 is directly and negatively regulated by miR-126-3p at both the post-transcriptional and translation levels, only translation levels negative TSC1 expression regulation.3. Annexin V-FITC and Edu detection showed that the inhibition PIK3R2 and TSC1(transfection of siRNA-PIK3R2 and siRNA-TSC1) can inhibit apoptosis of granulosa cells, promote granulosa cell proliferation. Supplementary exogenous siRNA-PIK3R2 and siRNA-TSC1 may be able to weaken promote granulosa cell apoptosis,which is due to inhibition miR-126-3p and reply due to inhibition of miR-126-3p induced inhibition of granule cell proliferation.4. Through the q RT-PCR detection of transfected with siRNA-PIK3R2 and siRNA-TSC1 where PI3 K pathway downstream gene expression changes. Test results show that transfection of siRNA-PIK3R2 make IGF1 R, INRS, PDK1 and AKT1 expression decreased, while the expression of IRS1 rise; PTEN expression did not obvious. After transfection of siRNA-TSC1 makes PTEN, AKT1, TSC2, rp S6 and m TOR expression increased, while PDK1 expression did not change significantly.These results reveal miR-126-3p can inhibit porcine ovarian granulosa apoptosis and promote granulosa cell proliferation by PIK3R2 and TSC1. Provide a reference for in-depth understanding of miRNA regulation of ovarian follicle development, but also for the follow-up Study PIK3-AKT pathway in ovarian granulosa cells of the foundation.