MIR-1275 Regulates Synthesis Of Estrogen And Apoptosis Of Porcine Ovarian Granulosa Cells By Targeting NR5A2

In female animals, each reproductive cycle will have numerous ovarian follicles growing in the ovary, but more than 99% of the follicles exist atresia phenomenon before ovulation, and the essence of follicular atresia is granulosa cells apoptosis. Therefore, study on apoptosis of granulosa cells has important theoretical significance on improving female reproductive performance and prolonging the period of reproduction for female animals. miRNA is a type of single chain,non-coding RNA whose general length is 19-25bp.Recent studies have showed that miRNA plays an important role in animal ovarian granulosa cells proliferation, apoptosis and differentiation, but studies on the regulation mechanism of miRAN on porcine granulosa cells apoptosis are rare. This study found that the expression of miR-1275 was upregulated during the porcine follicular atresia, while we analyze the miRNA expression in the process of follicular atresia. In order to further study about the function and regulation mechanism of miR-1275, in this study, porcine granulosa cells were the research object and we want to analyze its function in porcine granulosa cells apoptosis and estrogen synthesis. The results of the function of miR-1275 revealed in this study were important in elucidating the molecular mechanism of porcine granulosa cells apoptosis and follicular atresia. The main results of this thesis are as follows:1. Using gene chip technology we have studied the expression of miR-1275 in different porcine follicles during follicular atresia. The results indicated that miR-1275 is involved in the process of porcine follicular atresia.Using flow cytometry we found that granulosa cells apoptosis significantly increased (P<0.01), indicating that miR-1275 can promote porcine granulosa cells apoptosis. Using radioimmunoassay technique detected the concentration of estrogen in liquid culture. Results showed that compared with NC group, miR-1275 can significantly inhibit the synthesis of estrogen, and the difference was significant (P<0.05). Using Real-time PCR and Western Blot technology detected the expression levels of CYP19A1、CYP11A1 in porcine granulosa cells after transfected with miR-1275. The results suggested that the mRNA and protein level of CYP19A1 were decreased compared with NC group, the difference was highly significant(P<0.01); the mRNA and protein level of CYP11A1 were also decreased significantly, the difference was significant (P<0.05). These results indicated that miR-1275 can inhibit the expression of CYP19A1、CYP11A1 gene in porcine granulosa cells and estrogen production, promoting porcine granulosa cells apoptosis and follicular atresia.2. Bioinformatic results indicated that CYP19A1 and CYP11A1 were not the target gene of miR-1275, but NR5A2 is a candidate target gene of miR-1275. Transfect NR5A-3’UTR report vector and miR-1275 into 293 cells. Luciferase reporter system verified that NR5A2 is a target gene miR-1275. Then we transfected miR-1275 into porcine granulosa cells, Real-time PCR and Western Blot found that the mRNA and protein level of NR5A2 in porcine granulosa cells decreased significantly (P<0.01). Apoptosis of porcine granulosa cells was significantly decreased (P<0.05) after transfected with NR5A2 overexpression vector, and the concentration of estrogen was increased significantly (P<0.05). Results indicated that in order to promote the apoptosis of porcine granulosa cells, miR-1275 degradated the mRNA of NR5A2 and inhibited the synthesis of estrogen mainly through targeting 3’UTR of NR5A2 gene.3. Transfect the constructed NR5A2 expression vector into porcine granulosa cells. Real-time PCR and Western Blot results showed that mRNA and protein level of CYP19A1 were significantly increased (P<0.01); the mRNA and protein level of CYP11A1 were significantly increased (P<0.05) after transfected with NR5A2 overexpression vector. Luciferase reporter system analysis showed that NR5A2 increased CYP19A1、CYP11A1 promoter significantly (P<0.01).These results indicated that NR5A2 regulates CYP19A1^ CYP11A1 gene expression through regulating their promoter activity in porcine granulosa cells.