The Study Of The Effect Of MiR-196a-5p On Ovarian Granulosa Cell Function

Such environmental factors,such as high temperature in summer,have great harm to the reproductive performance of female animals.Therefore,it is of great significance to deeply understand the influencing mechanism,which is important for improving the reproductive performance of female animals and promoting the development of animal husbandry.As an important somatic cell,ovarian granulosa cells play important role in follicle development.As an important non-coding small molecule RNA,miRNA has been widely studied in recent years.In this study,based on the live heat stress model of Tianfu black rabbit constructed by our research group,significantly down-regulated miR-196a-5p was screened in the miRNA expression profiles of ovarian tissue in the heat stress group,and its function was verified in the ovarian granulosa cells of the model animal mouse.The main results are as follows:(1)By comparing miRNA expression profiles of ovarian tissues of Tianfu black rabbits in heat stress and non-heat stress groups,it was screened that miR-196a-5p was significantly down-regulated in ovarian tissues under heat stress(p<0.01).(2)miR-196a-5p mimics and mimics NC were transfected into mouse ovarian granulosa cells,respectively.m RNA expression profiles of the two groups were obtained by RNA-seq technology.A total of 102 differentially expressed genes were identified in the two groups.Compared with the control group,the mimics group had 49 up-regulated genes and 53 down-regulated genes.GO enrichment analysis showed that differentially expressed genes were mainly related with the regulatory processes of cell proliferation,growth,cell connectivity,transposon activity,and protein complex formation.KEGG enrichment analysis showed that differentially expressed genes were enriched in Gn RH secretion and regulation,cell adhesion,thyroid hormone signaling pathway,MAPK signaling pathway,oxytocin signaling pathway.(3)miR-196a-5p mimics,mimic NC,miR-196a-5p inhibitor and inhibitor NC were transfected into mouse ovarian granulosa cells,respectively.PCNA,CDK2,CDK4 and CCND2 relative expression of each group were detected at 24 h,30h and 36 h.The results showed that overexpression of miR-196a-5p could significantly up-regulate the expression of these genes(p<0.05).The opposite effect was observed when miR-196a-5p was inhibited(p<0.05).The results of Western blotting test were consistent with the above quantitative results.The results of Ed U assay showed that overexpression of miR-196a-5p could significantly increase the rate of cells proliferation(p<0.05),while inhibition of miR-196a-5p expression inhibit the cell proliferation(p<0.05).Flow cytometry showed that the proportion of cells in the G0/G1 phase was slightly lower in the group overexpressing miR-196a-5p,while the proportion of cells in the S phase was slightly higher.On the contrary,the proportion of cells in G0/G1 phase in inhibitor group was significantly higher than that in control group(p<0.05),and the proportion of cells in S phase was significantly lower than that in control group(p<0.05).These results indicate that miR-196a-5p can significantly promote the proliferation of ovarian granulosa cells.(4)miR-196a-5p mimics,mimics NC,miR-196a-5p inhibitor and inhibitor NC were transfected into mouse ovarian granulosa cells,respectively.The relative expression levels of apoptosis-related genes Bcl2,Bax,Caspase3 and Caspase9 were detected,and the results showed that there were no significant differences in the expression levels of the above marker genes among all groups.The apoptosis rate was detected by flow cytometry,and the results showed that there was no significant difference of the apoptosis rate among all groups.These results indicated that miR-196-5p had no significant effect on ovarian granulosa cell apoptosis.(5)miR-196a-5p mimics,mimic NC,miR-196a-5p inhibitor and inhibitor NC were transfected into mouse ovarian granulosa cells,respectively,and the expression of STAR gene related to steroid hormone secretion was detected.The results showed that overexpression of miR-196a-5p could significantly promote the expression of STAR at 30h(p<0.01),while inhibition of miR-196a-5p showed the opposite situation(p<0.01).The contents of E2 and P4 in the supernatant of cell culture were further detected by ELISA kit,and it was found that overexpression of miR-196a-5p could significantly increase the concentration of E2/P4(p<0.05).(6)It was predicted that ZMYND11 was a target gene of miR-196a-5p using Target Scan,and the results of double luciferase reporting system showed that there was an obvious targeting relationship between them.si RNA was transfected into ovarian granulosa cells to interfere the expression of ZMYND11 and the relative expression was quantified.The results showed that compared with the control group,the expression levels of cell cycle and steroid hormone-related genes,were increased in granulosa cells,especially PCNA,CCND2 and STAR(p<0.05).These results indicated that ZMYND11 gene could inhibit cell proliferation and hormone secretion.In summary,the effects of miR-196a-5p on ovarian granulosa cells are mainly reflected in the regulatory process of cell proliferation,growth,cell connectivity,transposon activity and protein complex formation.Moreover,miR-196a-5p can promote granulosa cell proliferation and hormone secretion by inhibiting ZMYND11 expression,and has no obvious effect on apoptosis.This study provides theoretical basis for deeply understanding of the molecular function and influencing mechanism of miR-196a-5p and provides theoretical guidance for the development of molecular breeding.