Effect And Mechanism Of Knockdown PERK On Apoptosis And Autophagy Induced By Sorafenib In Hepatoma HepG2 Cells

Objectives Sorafenib is a multi-kinase inhibitor shown to have survival benefits in advanced HCC.However,it confers only modest benefit in median overall survival,the resistance problem dramatically limited the usage of sorafenib in HCC with uncertain mechanism.Previous studies have documented sorafenib regulates autophagy and apoptosis in hepatoma cells associated with different unfolded protein response(UPR).The present study was designed to clarify the role of PERK in sorafenib induces autophagy and apoptosis in HepG2 cells.Methods Firstly,stable HepG2 cell lines of PERK knockdown was established with sgRNA-PERK lentivirus,the expression of PERK was verified by western blot and qRT-PCR.Trypen blue assay was then applied to investigate the effect of PERK knockdown,and sorafenib(10μM)induced-lethality in these established HepG2 cell lines in presence or absence of thapsigargin(Tg,500 nM).RT-PCR was employed to detect the PERK mRNA,CHOP mRNA.Western blot was further performed to analyze the protein expression of PERK,caspase3 and P62.Finally,the numbers of apoptotic cells in these cells were measured by flow cytometry.Results After PERK silencing,the expression of PERK protein and mRNA were significantly lower in sgRNA-PERK HepG2 cells vs NC HepG2 cells.Trypan blue assay found that the sorafenib alone and sorafenib combined with Tg induced-cell death was substantially decreased with about 8.5%and 15%in PERK silencing cells.RT-PCR found that knockdown PERK significantly decreased the expression of CHOP mRNA.In PERK silencing cells,the expression of PERK,pro-caspase3 protein were decreased and apoptotic numbers were dramatically inhibited.Interestingly,PERK knockdown obviously inhibited autophagy with increment in P62 in HepG2 cells in response to sorafenib or sorafenib plus Tg.Conclusions Taken together,these results suggested that PERK,one of the hallmarks of ER stress signaling,might cause cell-death autophagy and thereby contribute to enhance sorafenib derived apoptosis in hepatoma HepG2 cells,which may provide a potential critical target for overcoming the resistance of sorafenib in HCC in the future.