The Role Of MiR-1307/BRCA1 Axis In Porcine Ovarian Granulosa Cell Apoptosis

Breast cancer associated gene 1(BRCA1)is an important tumor suppressor,which is widely involved in many biological processes such as homologous recombination repair,checkpoint activation,DNA damage repair,protein ubiquitination,transcriptional regulation,chromatin remodeling,cell apoptosis and cell cycle arrest.Our previous studies demonstrated that BRCA1 gene is a candidate gene of the differential selection region between high and low fertility pig breeds,by using a specific-locus amplified fragment sequencing,indicating BRCA1 may be involved in sow fertility.However,the role of BRCA1 gene in sow reproduction is unclear.In this study,real-time PCR and immunohistochemistry were used to analyze the tissue expression pattern ovarian cellular expression profile of porcine BRCA1 gene;RNA interference technology was used to verify the role of BRCA1 in the apoptosis of porcine ovarian granulosa cells;PCR technology was used to clone the 3’-UTR sequence of BRCA1 gene;bioinformatics method was used to identify the RNA regulatory elements such as miRNA response element(MREs)on 3’-UTR;and investigated the role of miR-1307 in regulating BRCA1 expression and function in porcine granulosa cells.Our research results provide a basis for understanding the role of BRCA1 in sow reproduction.The results of this study are as follows:(1)Tissue and cell expression pattern of porcine BRCA1 geneRT-PCR technology was used to detect the expression of BRCA1 gene in pig heart,liver,spleen,lung,kidney,duodenum and ovary tissues.The results showed that BRCA1 mRNA was expressed in all 7 tissues,and highly expressed in lung the the ovary tissues,indicating that the porcine BRCA1 gene is a widely expressed gene and ovarian highly expressed gene.Immunohistochemical analysis showed that BRCA1 protein expression was detected in granulosa cells at all stages of follicular development.Real-time PCR and western blot showed that the expression level of BRCA1 mRNA and protein in healthy follicles was significantly higher than that in atretic follicles,suggesting that BRCA1 gene might be related to atretic follicles of porcine ovary.(2)The role of BRCA1 gene in the apoptosis of porcine ovarian granulosa cellsOvarian granulosa cell apoptosis is the main cause of follicular atresia.Real-time PCR and western blot found that mRNA and protein levels of BRCA1 were decreased significantly in BRCA 1-siRNA-treated porcine ovarian granulosa cells cultured in vitro.Flow cytometry showed that knockdown of BRCA1 significant increase the apoptosis rate of porcine granulosa cells and the level of pro-apoptotic gene BAX mRNA,and significant decrease the level of anti-apoptotic gene BCL-2 mRNA and BCL-2/BAX ratio(P<0.05),indicating that knockdown of BRCA1 can promote the apoptosis of porcine ovarian granulosa cells.Cell cycle analysis showed that the percentage of cells at S phase decreased significantly,while the percentage of cells at G2 phase increased significantly in BRCA1-silencing granulosa cells,indicating that inhibition of BRCA1 resulted in G2/M phase arrest of porcine ovarian granulosa cells.These results indicate that BRCA1 can regulate apoptosis and follicular atresia of porcine ovarian granulosa cells by influencing cell cycle process.(3)miR-1307 is a regulator targets BRCA1 gene in porcine granulosa cellsTo understand the regulatory mechanism of low expression of BRCA1 in atretic follicles,the 3’-UTR partial sequence of porcine BRCA1 gene was obtained by PCR amplification,cloning and sequencing,with a length of 1099 bp.Multiple AU-rich elements(ARE)and potential miRNA response elements(MREs)were detected in this region.Among these potential regulatory miRNAs,miR-1307 is a miRNA that its seed sequence that can fully bind to the 3’-UTR of porcine BRCA1 gene.Luciferase activity analysis showed that overexpression of miR-1307 can significantly inhibit the luciferase activity of BRCA1 3’-UTR report vector,but had no significant effect on the luciferase activity of the report vector of MRE mutation,indicating that miR-1307 can directly bind to the 3’-UTR of BRCA1 gene.Real-time PCR and western blot showed that miR-1307 significantly inhibit mRNA and protein levels of BRCA1 in porcine ovarian granulosa cells.These results suggest that BRCA1 is a direct target gene of miR-1307 in porcine granulosa cells.(4)miR-1307 regulation of porcine granulosa cell apoptosis by targeting BRCA1Porcine ovarian granulosa cells were cultured in vitro and transfected with miR-1307 mimics overexpressing miR-1307,flow cytometry showed that the apoptosis rate was significantly increased.Real-time PCR showed that the pro-apoptotic gene BAX mRNA level was up-regulated,BCL-2 mRNA level and BCL-2/BAX ratio were down-regulated.On the contrary,the apoptosis rate was significantly reduced,BAX mRNA level was significantly reduced,and BCL-2 mRNA level and BCL-2/BAX ratio increased significantly in miR-1307-inhibiting granulosa cells.These indicate that miR-1307 can promote the apoptosis of porcine ovarian granulosa cells.In addition,BRCA1-siRNA and miR-1307 inhibitor were co-transformed into porcine ovarian granulosa cells,and it was found that knocking down BRCA1 can reduce the inhibitory effect of miR-1307 inhibitors on apoptosis,indicating that miR-1307 can promote granulosa cell apoptosis of porcine ovaries by targeting BRCA1 gene.In summary,we showed that BRCA1 is a gene that highly expressed in ovarian tissue and is involved in porcine follicular atresia.We also demonstrated that BRCA1 inhibits the apoptosis of porcine ovarian granulosa cells and follicular atresia by affecting the cell cycle process.We identified miR-1307 as a functional regulatory miRNA targeting BRCA1 gene in porcine ovarian granulosa cells.