| Apoptosis,also known as programmed death,is the process of maintaining the balance of body's environment and discharging unnecessary harmful cells from the body.In the development process of mammalian ovarian follicles,all ovarian follicles have to go through the process of degradation and atresia,only a few follicles attain maturity.Granulosa cells play a key role in follicular development and are closely related to follicular atresia and other processes.Kisspeptins(Kp),a peptide hormone encoded by the KiSS-1 gene,acts through G protein-couple receptor-54(GPR54).Kp affects follitropin and luteinizing hormone secretion by regulating hypothalamic GnRH secretion.It is expressed in the hypothalamus,liver,pancreas,placenta,ovarian and other tissues.Our previous studies found that Kp was expressed in bovine ovarian granulosa cells and promoted the apoptosis of bovine granulosa cells,but its regulatory mechanism was not yet clearly known.In this study,100 nM kp-10 was used to treat bovine granulosa cells for 24 h.miRNA-mRNA sequencing correlation analysis was used to to filter miRNA and its target genes that promote apoptosis of bovine granulosa cells by kisspeptin.According to the combined results of miRNA-mRNA analysis,114 differentially expressed mRNAs,61 differentially expressed miRNAs and 2438 miRNA-mRNAs with different GO enrichment functions were obtained,and 9 distinct KEGG signaling pathways were enriched to the extent of being involved in the process of cell proliferation and apoptosis.Real-time quantitative PCR was used to verify the expression of differentially expressed miRNA and mRNA.The results showed that the gene expression results were the same as those of the miRNA-mRNA combined sequencing technology,demonstrating the accuracy of high-throughput results.According to the results of miRNA-mRNA correlation analysis,miR-27 b and SFRP2 may be involved in the role of Kp in promoting the apoptosis of bovine granulosa cells.The target relationship between miR-27 b and SFRP2 was identified by using the double fluorescent enzyme study.The recombinant and mutant vectors of the SFRP2 Gene 3'-UTR were successfully constructed,and using psiCHECK-2 plasmid,psicheck-2-sfrp2-3'-UTR and psicheck-2-mut-sfrp2-3'-UTR recombinant plasmids were constructed and successfully transfected to 293 T cells.By detecting the vigor ratio of firefly and sea-kidney type fluorescein enzyme,the target relationship between SFRP2 and miR-27 b was clarified.miR-27 b could be combined with sfrp2-3'UTR-end target sites and inhibit the expression of SFRP2 after transcription,confirming that SFRP2 was the target gene of miR-27 b.The relationship between miR-27 b and its target gene SFRP2 in regulating bovine granulosa cell apoptosis by Kisspeptin was studied by flow cytometry,Real-time quantitative PCR and cell transfection technique.The mechanism of kisspeptin in regulating bovine granulosa cell apoptosis was revealed.The results showed that miR-27 b mimic could promote the proliferation of granulosa cells and inhibit their apoptosis,and miR-27 b inhibitor could promote the apoptosis of bovine granulosa cells(p<0.05),suggesting that miR-27 b could inhibit the apoptosis of bovine granulosa cells.Conjunct effect of miR-27 b mimic and kp-10 could promote the apoptosis of bovine granulosa cells,and conjunct effect of miR-27 b inhibitor and kp-10 could significantly promote the apoptosis of bovine granulosa cells.Flow cytometry showed that SFRP2 siRNA significantly inhibited the apoptosis of bovine granulosa cells,the expression of apoptosis-inducing gene caspase-3 significantly decreased,and the expression of apoptotic gene Bcl-2 gene significantly increased.Conjunct effect of miR-27 b mimic and SFRP2 siRNA inhibited granulosa cell apoptosis,and conjunct effect of SFRP2 siRNA and miR-27 b inhibitor significantly promotied the apoptosis of bovine granulosa cells(p<0.05).The results of this study indicate that Kp promotes the apoptosis of bovine granulosa cells by miR-27 b targeting SFRP2 gene expression.The results not only reveal the molecular mechanism of kisspeptin in regulating bovine granulosa cell apoptosis,but also enrich the regulation network of bovine granulosa cell apoptosis. |
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