Researches On The Roles Of ATF4 In Endoplasmic Reticulum Stress-induced Mitochondria Unfolded Protein Response In Glioma Cells

Aims:This study is aimed to explore the roles of ATF4 in mitochondrial unfolding response(UPR^mt)under endoplasmic reticulum stress?ERS?in glioma,revealing the close relationship between ERS and UPR^mt in tumor cells and providing significant evidences for the intervention of organelle interactions to treat tumors.Methods:In this study,glioma cells U87 and SHG44 were used as research objects.The ERS inducers tunicamycin?Tm?and thapsigargin?Tg?were used to induce ERS in glioma cells.Gene interference was used to overexpress or knock down ATF4expression.Then the changes of UPR^mt associated proteins,mtDNA copy numbers,mitochondrial membrane potential,mitochondrial morphology and numbers were detected by molecular biological experimental techniques,such as Western blot,RT-qPCR,flow cytometry and mitotracker stain.Result:1.ERS can induce mitochondrial dysfunction in glioma cells.U87 and SHG44 cells were treated with Tm and Tg to construct ERS cell models.We tested the cell viability changes of U87 and SHG44 cells treated with Tm and Tg for24h by MTT,and selected the drug concentrations as cell viability at 80%,70%and 60%for low,medium and high ERS intensity.Western blots were used to detect ERS-related proteins BIP and ATF4 expression.which confirmed that the ERS cell models were successfully established.The molecular biology experimental techniques such as mitotracker,RT-qPCR,and flow cytometry were used to detect the changes of mtDNA copy number,and mitochondrial membrane potential.There was no significant change about mitochondrial morphology and mitochondrial membrane potential in low and medium concentration group.But in the high concentration group,the mitochondrial morphology became smaller?rounder and showed increasing number,and mitochondrial membrane potential decreased significantly.The mtDNA copy number decreased significantly in the medium and high concentration group.It can be seen that ERS can induce mitochondrial dysfunction in glioma cells.2.ERS can induce UPR^mt in glioma cells.In order to study the effect of ERS on UPR^mt,the expression of UPR^mt associated proteins,such as chaperones HSP60,HSP10,and mitochondrial protease CLPP,LON were detected by western blots after U87and SHG44 cells treated with Tm and Tg for 6?12h.We found that the expressions of HSP60 and HSP10 in U87 and SHG44cells treated with Tm for 6 and 12 hours were significantly increased in the low concentration drug group,and gradually decreased in the medium?high concentration drug group,while CLPP and LON increased gradually with the increase of drug concentration.After Tg treatment of U87 and SHG44 cells for6,12h,except that the expression of HSP60 and HSP10increased gradually with the increase of drug concentration at 6h,the other proteins changed the same as those of Tm treatment.We used RT-qPCR to detect the changes of UPR^mtt associated genes in U87 and SHG44 cells treated with Tm for6h in the mRNA levels,and found that these were consistent with the changes of proteins.Mitochondria was extracted to detect the UPR^mt associated proteins after SHG44 cells were treated with Tm for 12h,which was found to be consistent with the change of total protein expression.To inhibited ERS,we treated SHG44 cells with ERS inhibitor 4-PBA,and it showed that mitochondrial chaperones and proteases decreased significantly.Evidences above shows that ERS can induce UPR^mtt in glioma cells.3.ATF4 is a key factor in ERS-induced UPR^mt.Since ATF4is an important molecule of ERS,it also plays an important role when mitochondria is impaired.To this end,we overexpressed or knocked down ATF4 and detected changes of UPR^mtt associated proteins expression in U87 and SHG44 cells treated with low concentration of ERS inducer by western bolts.We found that the BIP expression decreased,and the expression of HSP60,HSP10 and LON?CLPP increased in ATF4overexpressed group.And the result of using siRNA to knock down ATF4 was opposite.It was indicated that ATF4 is a key factor for ERS-induced UPR^mt.4.Under sever ERS,overexpressing ATF4 can protect mitochondrial function.In order to explore the effect of ATF4 to cell viability under ERS,we detected the change of cell viability of U87 and SHG44 cells treated with Tm for 12h after ATF4 overexpression.It was found that the cell viability significantly increased after ATF4 overexpressed in the high drug concentration group.To further explore the effect of ATF4 on mitochondrial function under ERS,after ATF4 was overexpressed,we treated U87 and SHG44 cells with high concentration of Tm for 12h,and detected the change of mtDNA copy number by RT-qPCR,found that the mtDNA copy number increased in the overexpression group.After ATF4 was overexpressed,SHG44 cells were treated with Tm at high concentration for 24h.Flow cytometry was used to detect changes of mitochondrial membrane potential.The mitochondrial membrane potential increased in ATF4overexpression group.It suggested that ATF4 overexpression could protect mitochondria under sever ERS.Conclusion:ATF4 is a key factor in ERS-induced UPR^mt and protects mitochondria from ERS in glioma cells.