The Effects Of CHOP Overexpression On HCC Proliferation,Apoptosis And Cdc25C

Objective:To investigate the relationship between CHOP,Cdc25C and HCC by studying the effects of CHOP overexpression on HCC proliferation,apoptosis and Cdc25C,in order to further study the mechanisms of Cdc25C in HCC development.Methods:(1)Construction of CHOP lentiviralvector(LV-CHOP)for transfection of mouse hepatoma Hepal-6 cells line as experimental group(LV-CHOP-Hepal-6).The mouse hepatoma Hepal-6 cells transfected with lentiviral vector were used as negative control group(LV-Hepal-6).The untransfected mouse hepatoma Hepal-6 cells were used as blank control group(Hepal-6).The expression of CHOP in mouse hepatoma Hepal-6 cells was detected by RT-PCR and Western blot methods,respectively.(2)The proliferation and migration ability of Hepal-6 cells in each group were detected by CCK-8 method and scratch repair test.(3)The cell cycle of each group of Hepal-6 cells was detected by flow cytometry.(4)Flow cytometry and Westerm blot were used to detect the apoptosis of Hepal-6 cells.(5)Western blot method was used to detect the expression of GRP78,the endoplasmic reticulum stress response marker,in mouse hepatoma Hepa1-6 cells.(6)The expression of Cdc25C mRNA in Hepa1-6 cells was detected by qRT-PCR method.The Cdc25C protein expression in Hepa1-6 cells was detected by Western blot and immunofluorescence staining methods,respectively.Results:(1)The LV-CHOP lentiviral vector was successfully constructed.After transfecting mouse hepatoma Hepa1-6 cells,the CHOP expression levels in experimental group was significantly higher than that of in the negative control group and the blank control group.The mouse hepatoma Hepa1-6 cells overexpressing CHOP were obtained.(2)The results of CCK-8 assay showed that CHOP overexpression inhibited the proliferation of mouse hepatoma Hepa1-6 cells.Compared with the control groups,the difference was statistically significant(P<0.01).(3)In the Wound-healing experiment at 12h,there was no significant difference in cell repair rate between the three groups of mouse hepatoma Hepa1-6 cells(P>0.05).But in the Wound-healing experiment at 24h,the cell repair rate of LV-CHOP-Hepa1-6 experimental group was lower than that of Hepa1-6 and LV-Hepa1-6 control group(P<0.01).(4)The results of cell cycle assay by flow cytometry showed that after up-regulation of CHOP,G0/G1 phase cells increased(P<0.05),and S phase cells decreased significant(P<0.01).But,there was no significant difference in G2/M phase cells(P>0.05).(5)Flow cytometry results showed that after up-regulation of CHOP,it could significantly proruote the apoptosis of mouse hepatoma Hepa1-6 cells(P<0.01).Western blot analysis showed that after up-regulation of CHOP,the expression of caspase3 and Bax proteins were increased,and the expression of anti-apoptotic protein Bcl-2 protein was decreased(P<0.05).(6)After CHOP overexpression,the GRP78 protein expression was increased(P<0.05).(7)qRT-PCR assay showed that the level of Cdc25C mRNA in mouse hepatoma Hepa1-6 cells of LV-CHOP-Hepa1-6 experimental group was lower than that of in the control groups(P<0.05).The results of Western blotting and immunofluorescence cytochemical staining showed that the Cdc25C protein expression in mouse hepatoma Hepa1-6 cells of LV-CHOP-Hepa1-6 experimental group was lower than that of in the control groups(P<0.05).Conclusion:Over expression of endoplasmic reticulum stress-specific transcription factor CHOP could attenuate the proliferation and migration of mouse hepatoma Hepa1-6 cells,promote apoptosis by inducing endoplasmic reticulum stress,and reduce the expression of Cdc25C.