Study On The Mechanism Of ZCL-082 Induced Autophagy Of Mouse Female Germline Stem Cells In Vitro

Background:Recent studies mainly focus on female germline stem cells(FGSCs)differentiation,proliferation,and apoptosis,but other biological processes such as autophagy and its mechanism in FGSCs are largely unclear.Besides,small molecule compounds play an important role in the study of the mechanism of autophagy in cells because of immediate and reversible effects.Benzoxaboroles have great potential in clinical research and treatment due to their good chemical properties.However,there is no studies have been reported on the role of benzoxaboroles in FGSCs yet.Purposes:To explore the function and regulation mechanism of ZCL-082 affects FGSCs.Results:In the present study,we added the compound ZCL-082(a derivative of a benzoborazole heterocycle)to FGSCs to examine its effect.Using a cell counting kit-8(CCK8)and 5-ethynyl-2'-deoxyuridine(EdU)assays,we found that ZCL-082 could significantly reduce the viability,proliferation,and number of FGSCs in vitro.Moreover,western blotting revealed that the expression of light chain 3 beta 2(LC3BII)in FGSCs was significantly increased after treatment with ZCL-082 for 3 h and 6 h.Meanwhile,the expression of sequestosome-1(SQSTM1)was significantly decreased.These results suggested that ZCL-082 can induce autophagy of FGSCs in vitro.Regarding the molecular mechanism,ZCL-082 could significantly reduce the expression of growth arrest-specific 5(GAS5)long non-coding RNA,which could directly bind to microRNA-21a(miR-21a)and negatively regulate each other in FGSCs.Knockdown of GAS5 induced autophagy of FGSCs,while GAS5 overexpression inhibited autophagy of FGSCs in vitro and rescued FGSCS autophagy induced by ZCL-082.Additionally,overexpression of miR-21 a significantly enhanced LC3B-II protein expression,while significantly reducing the expression of programmed cell death protein 4(PDCD4)and SQSTM1 protein in FGSCs compared with control cells.Inhibition of miR-21 a significantly reduced the basal or ZCL-082-induced upregulated expression of LC3B-II and significantly enhanced the expression of PDCD4,while downregulating the basal or ZCL-082-induced expression of SQSTM1 in FGSCs.Conclusions:This study showed that ZCL-082 can reduce the viability and proliferation and induce apoptosis and autophagy of FGSCs in vitro.Overexpression of GAS5 rescue autophagy of FGSCs induced by ZCL-082;ZCL-082 regulates autophagy of FGSCs by GAS5/miR-21 a axis.These results are helpful to further understand the physiological functions and developmental mechanism of FGSCs,and also provide new sight for maintaining female fertility and clinical treatment of reproductive disorders.