Mechanism Of CircAKT3 Affect The Self-Renewal Of Female Germline Stem Cells

Objective Circular RNAs(circ RNAs)are a class of noncoding RNAs with covalently closed circular structures.They are structurally stable,highly conserved and specifically expressed during tissue development.Circ RNAs are involved in a variety of biological processes,such as miRNA sponge adsorption function.A large number of studies have shown that circ RNAs are closely related to stem cells and reproductive development.Female germline stem cells(FGSCs)are adult stem cells that can both self-renew and differentiate into mature oocytes.However,there are few reports on the regulation of FGSCs by circ RNAs.Therefore,exploring their functions in the development of FGSCs will help us to understand the regulation mechanism of germ cell development,and add new evidence for the mechanism of non-coding RNA and improve the quality of oocytes To provide a theoretical basis for the diagnosis and treatment of nest dysfunction and other related diseases.Methods FGSCs were identified by RT-PCR and cell immunofluorescence;reverse splicing of circ AKT3 was identified by RT-PCR;covalent closure of circakt3 was verified by RNase R;cyclization site of circakt3 was verified by Sanger sequencing;subcellular localization of circakt3 in FGSCs was verified by fluorescence in situ hybridization(FISH);the effect of circ AKT3 on FGSCs activity and proliferation was detected by CCK-8 and Ed U experiments;annexin The effects of circ AKT3 on the apoptosis of FGSCs were detected by v-pe method;the miRNAs that may interact with circ AKT3 were predicted by Miranda and verified by RT-q PCR;the interaction of circ AKT3 with mir-9-5p and 3 'UTR of mir-9-5p and Akt3 was verified by dual luciferase assay;the interaction of circ AKT3 with mir-9-5p and 3'UTR of mir-9-5p and Akt3 was further verified by RNA immunoprecipitation assay(RIP).Results Overexpression of circ AKT3 in FGSCs can significantly promote the proliferation of FGSCs.Meanwhile,knockdown of circ AKT3 could inhibit the proliferation of FGSCs without inducing apoptosis.Luciferase Report and rip experiment confirmed the interaction between circakt3 and miR-9-5p,and Akt3 3 'UTR and miR-9-5p.RT q PCR results showed that overexpression of circ AKT3 significantly increased the expression of Akt3,whereas knockdown of circ AKT3 significantly decreased the expression of Akt3.Conclusion In FGSCs,circ AKT3 acts as a miRNA molecular sponge to adsorb miR-9-5p,which can release the inhibition of its target gene Akt3 and promote FGSCs self-renewal;while knockdown of circ AKT3 can release miR-9-5p,which can inhibit the expression of its target gene Akt3 and inhibit FGSCs self-renewal.This study revealed the role of circ AKT3 in the development of FGSCs,and provided a basis for the follow-up study of circ RNAs regulating the development of FGSCs,thus providing a new perspective for the prevention and treatment of female infertility and other diseases.