Regulation And Mechanism Of Secretagogin In Ox-LDL-induced Islet ? Cells Endoplasmic Reticulum Stress

Objective To investigate the role of secretagogin(SCGN)in regulating oxidized-low density lipoprotein(ox-LDL)induced endoplasmic reticulum stress and associated apoptosis in islet ? cells.Methods1.Mouse islet derived MIN6 cells were treated with increasing concentrations of ox-LDL,and then Oil Red O staining,BODIPY staining,CCK8 and flow cytometry assays were performed to detect the intracellular lipid content,cell viability and cell apoptosis of MIN6 cells,respectively.2.Western blot(WB)was used to detect the expression levels of SCGN,endoplasmic reticulum(ER)stress-related proteins(PERK,p-PERK,p-eIF2?,eIF2?,ATF4,CHOP)and apoptosis-related protein Cleaved Caspase3.3.For gene-silencing studies,MIN6 cells were transfected with siRNAs against SCGN or scrambled siRNA and maintained for 24 hours.Next,the expression of SCGN,PERK,p-PERK,p-eIF2?,eIF2?,ATF4 and Cleaved Caspase3 were measured in cells treated with SCGN specific siRNA or 40?g/ml of ox-LDL.4.To restore the function of SCGN in ox-LDL treated MIN6 cells and further clarify the effect of SCGN on endoplasmic reticulum stress and apoptosis in MIN6 cells,a recombinant plasmid harboring the full cDNA sequences of SCGN(rSCGN)was constructed and transfected into MIN6 cells,and then cells expressing SCGN stably were established.Flow cytometry analysis was used to detect the cell apoptosis rate and WB assay was applied to measure the ER stress-related proteins in cells treated with ox-LDL alone,ox-LDL in combination with rSCGN,ox-LDL in combination with sodium 4-phenylbutyrate(PBA)or ox-LDL in combination with rSCGN and PBA,.5.An integrated bioinformatical analysis was conducted to predict the interaction and potential binding sites between ATF4 and SCGN.Next,immunoprecipitation method(CO-IP)assay was implemented to confirm the interaction between these two proteins.Results1.Oil Red O and BODIPY assay showed that with the increase of ox-LDL concentration,the intracellular lipid accumulation in MIN6 cells was increased.Meanwhile,the cell apoptosis rate was elevated,while the cell viability was decreased.2.The expression of SCGN was significantly down-regulated,whilethe expression of p-PERK,p-eIF2?,ATF4,CHOP and Cleaved Caspase3 was significantly increased in MIN6 cells treated with ox-LDL for 24 hours.3.SCGN-siRNA promotes the expression of ATF4,CHOP and Cleaved Caspase3,but it has no effect on the expression of PERK,p-PERK,eIF2? and p-eIF2?,suggesting that SCGN affects endoplasmic reticulum stress in MIN6 cells by regulating ATF4/CHOP/Cleaved Caspase3 signal pathway.4.Flow cytometry and WB experiments showed that rSCGN or PBA pretreatment could partially inhibit ox-LDL-induced endoplasmic reticulum stress and apoptosis in MIN6 cells,while rSCGN combined with PBA treatment could achieve a better inhibitory effect than SCGN or PBA alone.These results suggest that SCGN/ATF4 and PERK/eIF2?/ATF4 pathway are involved in the regulation of ox-LDL-induced endoplasmic reticulum stress in MIN6 cells.5.Bioinformatics analysis and CO-IP experiments confirmed the interaction between SCGN and ATF4 in MIN6 cells.Conclusions1.SCGN can bind to ATF4 and regulate ATF4 expression negatively in islet ? cells.2.SCGN/ATF4 signal pathway up-regulates ox-LDL-induced endoplasmic reticulum stress and apoptosis in islet ? cells.