Molecular Mechanism Of CircR-PANK2 Regulating DNA Damage And Steroid Hormone Secretion In Bovine Cumulus Cells Via MiR-302d/CDKN1A Pathway

Cumulus cells,as a group of granulosa cells surrounding oocytes,provide nutrients to oocytes and transmit important biological information through various communication methods.Their normal physiological functions are crucial to ensure the normal communication mechanism between cells,the quality of oocytes and the development of embryos.DNA damage in cumulus cells,represented by DNA double strand break(DSB),can destroy genome stability,induce apoptosis,abnormal steroid hormone secretion,and then lead to meiosis block and maturation quality decline of oocytes,seriously affecting the reproductive ability of animals.However,the key signaling molecules that regulate the development of DSB in cumulus cells and their mechanism of action are still unclear.Therefore,it is of great theoretical and practical value to reveal the key molecules in the intracellular response during DNA damage to improve the quality of oocyte maturation and embryo development potential.In response to external signal stimulation,non-codingRNA can regulate gene expression through post-transcription and affect cell physiological function.The specific expression of non-codingRNA in cumulus cells plays an important role in oocyte maturation and early embryonic development.However,the expression and mechanism of non-codingRNA during DNA damage in cumulus cells remain unclear.Therefore,clarifying the physiological function of non-codingRNA in cumulus cells and systematically analyzing the regulatory mechanism of non-codingRNA in the process of DNA damage on the cell function of cumulus cells have important research significance for solving follicular developmental disorders.In this study,DNA damage model of bovine cumulus oocyte was established by bleomycin treatment and whole transcriptomic analysis was performed to screen out CDKN1 A,a key factor that may be involved in regulating DNA damage,and its potential circRNA regulatory network.Through cell transfection,flow cytometry,RTq PCR,WB and ELISA experiments,the role of CDKN1 A in regulating cell proliferation and steroid hormone secretion during the DNA damage of bovine cumulus cells was confirmed.The molecular mechanism of circ R-PANK2 regulating DNA damage,proliferation,apoptosis and steroid hormone secretion of bovine cumulus cells through miR-302d/CDKN1 A pathway was further analyzed.Specific results are as follows:1.The DNA damage model of bovine cumulus oocyte was successfully established,and the positive rate of γH2AX cells was significantly increased,the expressions of DNA damage marker genes MRE11,ATM and RAD51 were increased,the expression of BRCA1 was decreased,the cell proliferation was inhibited,the cell cycle was disturbed,and the apoptosis rate was significantly increased.2.The whole transcriptome differential expression profile of bovine cumulus cells after DNA damage was successfully constructed,and 848 mRNA,72 miRNA,66 circRNA and 75 lncRNA were differentially expressed.The ceRNA regulatory network of circRNA-miRNA-mRNA was successfully constructed,and CDKN1 A,a key molecule regulating DNA damage,was screened out.3.After CDKN1 A knockdown in bovine cumulus cells,the expressions of DNA damage-related genes BRCA1,MRE11 and ATM were increased,the expression of RAD51 was decreased,and the rate of γH2AX positive cells was significantly reduced,which alleviated DNA damage.The expressions of CDK1,CDK2 and CCNE2 were increased,while the expression of CCND1 was decreased,which promoted the proliferation of cumulus cells.Apoptosis-related genes P53 and FAS were decreased,and the ratio of BAX to BCL2 was decreased,which inhibited apoptosis of cumulus cells.The expression of STAR,CYP11A1 and HSD3B1 genes related to steroid hormone secretion increased,which promoted the secretion of steroid hormone in cumulus cells.After overexpression of CDKN1 A,gene expression and cell phenotype related to DNA damage,cell proliferation,apoptosis and steroid hormone synthesis showed opposite trend compared with inhibition of CDKN1 A.4.miR-302 d can target CDKN1 A binding and inhibit its expression,resulting in corresponding changes in the expression of genes related to DNA damage,cell proliferation,apoptosis and steroid hormone secretion,which can alleviate DNA damage of cumulus cells,inhibit apoptosis of cumulus cells,promote proliferation of cumulus cells and increase the level of steroid hormone secretion of cumulus cells.5.circR-PANK2 formed by the cyclization of exon 3 to 6 of PANK2 gene acts as a molecular sponge of miR-302 d,targeting the binding sequence UUCGUGAA,relieving the post-transcriptional regulatory inhibition of CDNK1 A by miR-302 d,and regulating DNA damage and related physiological functions of cumulus cells.In summary,this study took the regulatory mechanism of competitive endogenousRNA(ceRNA)as an entry point to elucidate the regulatory effects of CDKN1 A,as a key factor regulating DNA damage,on proliferation,apoptosis and steroid hormone secretion of bovine cumulus cells.The molecular mechanism of circ R-PANK2 regulating DNA damage and steroid hormone secretion in bovine cumulus cells through miR-302d/CDKN1 A pathway was revealed.The regulation mechanism of DNA damage and steroid secretion in cumulus cells was elucidated from the epigenetic level,which provided a new way to maintain the stability of DNA in cumulus cells,and also provided a new research strategy for further interpreting the regulation mechanism of follicle development and improving the fertility of cows.