Studies On The Autophagy And The Mechanism Induced By Benzobonazole Compound 89 In Female Germline Stem Cells

Female germline stem cells(FGSCs)are a kind of germ cells that have the ability to self-renew and differentiate into oocytes.Some studies showed that autophagy is involved in mediating germ cell survival and death,but there is no report on the effect of autophagy on FGSCs and related molecular mechanisms.Benzoborazoles are widely used in anti-inflammatory,anti-bacterial and anti-cancer aspects,they are suitable for experimental research and clinical applications.In terms of cell function studies,benzoborazoles are only known to inhibit the growth of human ovarian cancer skov-3 cells,suggesting that they may induce cell autophagy or apoptosis.Compound 89(C89)is a kind of benzoborazoles.The aim of this study was to investigate the molecular mechanisms involved in the autophagy of FGSCs by studying the effects of C89 on FGSCs.After screening of several benzoxaboroles,it was found that C89 inhibited the growth of FGSCs.To investigate the effect of C89 on the development of FGSCs,after treated with C89(0.5,1,2 ?M),cytometry,Cell Counting Kit-8(CCK8)and 5-ethynyl-2-deoxyuridine(EdU)assay showed that the number,viability and proliferation of FGSCs were significantly reduced in C89-treated groups compared with controls(P < 0.01).Then the differentiation-related marker genes stra8 and sycp3 were detected by reverse transcription-polymerase chain reaction(RT-PCR).there is no expression of stra8 and sycp3 in C89-treated FGSCs.Flow cytometry showed no significant difference in apoptosis rates between C89-treated FGSCs and control groups.The autophagy-related marker proteins microtubule-associated protein light chain 3 beta II(LC3BII)and sequestosome-1(SQSTM1)were detected by Western blot.The results showed that the expression of LC3 BII in C89-treated FGSCs was significant increased compared with the control groups(P < 0.01),and SQSTM1 was significantly decreased(P < 0.001).This indicates that under the same conditions,C89 can not induce FGSC apoptosis or differentiation,but can induce FGSC autophagy.To investigate the mechanism of C89 induced FGSC autophagy,RNA-seq technique was used to compare the transcriptome differences between C89-treated FGSCs and controls.We screened 937 up-regulated and 1046 down-regulated differentially expressed mRNAs,and four differentially expressed mRNAs,Bcl2,Atg7,Trp53 and Rubcn,were related to autophagy.The expressions of these mRNAs were verified by quantitative real-time-polymerase chain reactionq(RT-PCR),and the qRT-PCR results were consistent with RNA-seq data.Additionally,the phosphatidylinositol 3 kinase and kinase Akt(PI3K-Akt)pathway may be involved in the effects of C89 in inducing autophagy in FGSCs.The expression levels of p-PI3 K and p-Akt were detected by Western blot,and the results confirmed that levels of p-PI3 K and p-Akt were significantly reduced in the C89-treated groups compared with controls(P < 0.05),which indicating that C89 inhibited the activity of the PI3K/Akt pathway.In addition,we also investigated the effect of PI3 K inhibitor LY294002 on FGSCs.The results showed the cooperative functions of C89 and LY294002 in inducing FGSC autophagy through suppressing the PI3K-Akt pathway.Taken together,this study demonstrates that C89 can induce FGSC autophagy by inhibiting the activity of PI3 K and Akt,thereby reducing the number,viability,and proliferation of FGSCs.The PI3K-Akt pathway may be a target to regulate FGSC autophagy,while C89 has the potential to be a novel inhibitor of the PI3K/Akt pathway.