Effect Of SPAG6 Gene Silencing On Autophagy Of SKM-1 Cells And Its Mechanism

Objective:To construct the pGC-shRNA-SPAG6(short hairpin RNA)lentivirus targeting silence SPAG6 gene in MDS cell line,and investigate the effect of silencing SPAG6 gene on autophagy and its underlying molecular mechanisms as well as the relationship between autophagy and apoptosis in SKM-1 cells.Methods:1.To screened out the optimal shRNA for SPAG6 and established the SPAG6-/-infected SKM-1 cell line: SKM-1 cells were infected with negative control lentiviral vector(NC-shRNA)or short hairpin RNA lentiviral vectors targeting SPAG6 gene expression(SPAG6-shRNAs).The green fluorescent protein(GFP)expression of SKM-1 cells was observed by fluorescence microscope and and the efficiency of lentivirus transfection was detected by flow cytometry.The optimal shRNA for SPAG6 was screened out by RT-qPCR and Western blot analysis.Then,to screen out the stable infected cell lines,0.5 μg / ml puromycin was added in the medium to culture the SKM-1 cells for 2 weeks.2.To explore the effect of SPAG6 silencing on autophagy andapoptosis in SKM-1 cells: After cells were infected with NC-shRNA or SPAG6-shRNA,the mRNA expression levels of autophagy-associated genes were detected by qRT-PCR;the autophagosomes formation of each group was observed by TEM;the expression levels of autophagy and apoptosis-associated proteins were detected by Western blot analysis;the apoptosis rate was detected by flow cytometry.3.To explore the possible relationship between autophagy and apoptosis in SKM-1 cells: After cells were co-treated with SPAG6-shRNA and autophagy inhibitors(3-MA and CQ),Western blot analysis was applied to detect the expression levels of autophagy and apoptosisassociated proteins;the apoptosis rate was detected by flow cytometry.4.To investigate the underlying molecular mechanisms of SPAG6 gene in regulating cell autophagy in SKM-1 cells: Western blot was used to detect the expression levels of AMPK / mTOR / ULK1 signaling pathway proteins.Subsequently,after SKM-1 cells were treated with AMPK inhibitor(Comound C),the expressions of AMPK / mTOR / ULK1 pathway proteins and autophagy and apoptosis-associated proteins were detected by Western blot analysis.The apoptosis rate was determined by flow cytometry.Results:1.Screened out the optimal shRNA for SPAG6 and established the SPAG6-/-infected SKM-1 cell lines successfully: After cells were infectedwith NC-shRNA and SPAG6-shRNAs,flow cytometry indicated that the efficiency of each group was more than 80%;The optimal shRNA for SPAG6 was screened out by RT-qPCR and western blot analysis,which shown as SPAG6-shRNA3.2.SPAG6 silencing induces autophagy and aopotosis in SKM-1 cells.Compared with NC-shRNA group,the mRNA expression levels of LC3-Ⅱ、Beclin-1、ATG-5、ATG-7 were increased in SKM-1 cells infected with SPAG6-shRNA.In addition,TEM showed that the number of autophagosome formation was higher in SPAG6-shRNA group than that in NC-shRNA group,and Western blot analysis yielded the same results;Moreover,Western blot analysis demonstrated that compared with SKM-1clles treated with NC-shRNA,the protein expression levels of Bax and cleaved caspase-3 were increased in the SKM-1 cells treated with SPAG6-shRNA,while the protein expression level of Bcl-2 was increased.Flow cytometry showed that the apoptosis rate in SPAG6-shRNA was higher than that in NC-shRNA.3.Inhibition of autophagy with autophagy inhibitors(3-MA and CQ)attenuates SPAG6 knockdown-induced apoptosis in SKM-1 cells: Western blot analysis demonstrated that compared with that in clles treated with SPAG6-shRNA alone,the expression levels of Bax and cleaved caspase-3were decreased but the expression level of Bcl-2 was increased in cells co-treated with SPAG6-shRNA and 3-MA/CQ.Flow cytometry showedthat the apoptosis rate in SPAG6-shRNA-3-MA/CQ group was lower than that in SPAG6-shRNA group.4.SPAG6 downregulation induces autophagy via the AMPK/mTOR/ULK1 pathway: Western blot analysis showed that although the total AMPK,ULK1 and mTOR levels were not altered at all,the expression levels of phosphorylated AMPK and ULK1 were remarkably increased by SPAG6 downregulation,while phosphorylated mTOR level was significantly decreased.Then,we co-treated SKM-1 cells with AMPK inhibitor(Compound C)and SPAG6-shRNA,which showed that compared with that in cells treated with SPAG6-shRNA alone,the expression levels of phosphorylated AMPK was decreased,accompanied by a decrease of the expression of LC3-Ⅱ、Beclin-1、Bax and cleave caspase-3,but an increase of the expression of P62 and Bcl-2.Flow cytometry showed that the apoptosis rate in cells cotreated with SPAG6-shRNA and Compound C was lower than that in cells treated with SPAG6-shRNA alone.Conclusion:Downregulation of SPAG6 triggers autophagy via upregulating the AMPK/mTOR/ULK1 signaling pathway,which further contributes to the apoptosis of SKM-1 cells induced by SPAG6 knockdown.Thus,our findings indicated that SPAG6 might be a potential therapeutic target against MDS and that autophagy might be considered a potential mechanism for the treatment of MDS.