MicroRNA-18b Regulates The Function Of Rabbit Granulosa Cells

The fertility of female mammals is closely related to the development and atresia of ovarian follicles.As an important part of follicles,granulosa cells are essential for the survival and growth of oocytes and the synthesis and secretion of steroids.As a negative regulator of post-transcriptional gene expression,miRNA regulates the function and apoptosis of granulosa cells by interacting with target m RNA.Rabbits are important economic animals and also good animal models,but there are few studies on the function of miRNAs in the development of rabbit ovaries.It is reported that miRNA(miR)-18 b which belongs to miR-106a-363 ??cluster plays an important role in the development of human reproductive system diseases.Therefore,this study will explore the expression of miR-18 b during steroid hormone synthesis and its effect on the proliferation and apoptosis of granulosa cells.In this study,rabbit granulosa cells were used as the research object.Through overexpression and inhibition of miR-18 b,enzyme-linked immunosorbent assay(ELISA),CCK8 cell proliferation detection method,flow cytometry and Annexin V-FITC / PI staining,real-time quantification Experimental techniques such as PCR(Real-time q PCR),dual luciferase and Western Blot(Western Blot)were used to explore the effect of miR-18 b on granulosa cell function.And the target gene of miR-18 b were predicted and verified.These results provide a new perspective for the in-depth study of the function and mechanism of regulating steroid hormones in rabbit granulosa cells.The results are as follows:1.The basic functions of miR-18 b is predicted by bioinformatics method.Besides,rabbit miR-18 b is highly conservative in evolution.Then,by predictng and analyzing the direct targets of miR-18 b,it was found that the signal pathways enriched for miR-18 b target genes are in cell proliferation and apoptosis,etc.Most of these results are consistent with signaling pathways which are known to play a role in ovarian development,maturation,and atresia.These provides a basis for further exploring the biological function of miR-18 b in ovarian granulosa cells.2.Firstly,this study isolated and identified rabbit granulosa cells.Then,CCK-8 method and flow cytometry were used to detect cell proliferation and cell cycle.The results showed that after overexpression of miR-18 b,the cell cycle stalled before the S phase,thereby inhibiting DNA synthesis and cell proliferation.The results of Annexin V-FITC fluorescencedetection showed that miR-18 b can promote the apoptosis of granulosa cells.Furtheremore,q PCR was used to detect the expression of apoptosis-related genes(Caspase-3,Bax and Bcl2),which was consistent with the results of apoptosis detection.3.Granulosa cells are the main sites for secreting estradiol(E2)and progesterone(P4).After overexpression of miR-18 b,the expression of E2 in the cell culture medium was reduced using ELISA detecting.But P4 synthesis was not significantly affected.And the expression of hormone synthesis related genes(CYP19A1,CYP11A1 and 17B-HSD1)was detected by q PCR.The results showed that the silencing of miR-18 b increased the expression of genes related to E2 production.4.In this experiment,the dual luciferase assay verified that miR-18 b directly targets the3'UTR region of ESR1.In rabbit granulosa cells,overexpression of miR-18 b resulted in a decrease in ESR1 m RNA and protein levels,which further proved that miR-18 b and ESR1 have a negative correlation expression pattern.The above results indicated that miR-18 b can regulate hormone secretion and influence rabbit ovarian granulosa cells apoptosis.The overexpression of miR-18 b can inhibit the proliferation of granulosa cells and promote cell apoptosis.At the same time,miR-18 b can act as a negative regulator to mediate the production of enzymes related to E2 synthesis and down-regulate the level of E2.Otherwise,this study also predicted and identified the direct target gene of miR-18 b,ESR1.The research results provide a new candidate gene for further understanding of miRNA affecting follicle development,which is of great significance for improving the economic benefits of livestock production.