QRICH1介导NF-κB p65调控Bcl-2、Bax在砷致肝癌细胞凋亡中的作用研究

The role of QRICH1 mediating NF-κB p65 in regulating Bcl-2 and Bax in arsenic-induced hepatocellular carcinoma cell apoptosis

目的:探讨砷(AS)诱导肝癌细胞凋亡过程中QRICH1通过调控NF-κB p65表达水平调控Bcl-2、Bax的作用机制。方法:体外培养人的肝癌细胞(HepG2细胞),采用慢病毒转染的方式,构建过表达/敲低QRICH1稳定表达HepG2细胞株。分为对照组、加砷组、砷+QRICH1过表达阴性对照组、砷+QRICH1过表达组、砷+QRICH1敲低阴性对照组、砷+QRICH1敲低组。在细胞对数生长期时,加入40μmol/L亚砷酸钠(NaAsO_(2))作为终浓度处理24 h。对照组加入与NaAsO_(2)同等体积的磷酸缓冲盐溶液(PBS)处理。采用细胞计数(CCK-8)检测细胞增殖情况;流式细胞术检测各组细胞凋亡情况;蛋白免疫印迹法(Western blot)检测各组细胞QRICH1、NF-κB p65及Bcl-2、Bax蛋白表达水平。结果:CCK-8结果显示与对照组比较,砷处理组增殖率比例明显降低(P<0.05);与砷+QRICH1过表达阴性对照组比较,砷+QRICH1过表达组细胞增殖率明显上升(P<0.05),与砷+QRICH1敲低阴性对照组比较,砷+QRICH1敲低组细胞增殖率明显下降(P<0.05),差异具有统计学意义。流式细胞术结果显示与对照组比较,砷处理组总凋亡率比例明显升高(P<0.05);与砷+QRICH1过表达阴性对照组比较,砷+QRICH1过表达组细胞总凋亡率比例明显下降(P<0.05),与砷+QRICH1敲低阴性对照组比较,砷+QRICH1敲低组细胞总凋亡率比例明显上升(P<0.05)。蛋白免疫印迹法结果显示与对照组比较,砷处理组QRICH1、NF-κB p65及Bcl-2蛋白表达水平较低,Bax的蛋白表达水平较高(P<0.05)。与砷+QRICH1过表达阴性对照组比较,砷+QRICH1过表达组QRICH1、NF-κB p65及Bcl-2蛋白表达水平较高,Bax的蛋白表达水平较低(P<0.05)。与砷+QRICH1敲低阴性对照组比较,砷+QRICH1敲低组QRICH1、NF-κB p65及Bcl-2蛋白表达水平较低,Bax的蛋白表达水平较高(P<0.05),差异具有统计学意义。结论:砷致HepG2凋亡过程中,QRICH1通过调节NF-κB p65的表达,进而影响了Bcl-2、Bax的表达。提示QRICH1可能是促进肝癌细胞凋亡的潜在作用靶点。

Objective:To explore the mechanism of QRICH1 regulating Bcl-2 and Bax by regulating the expression level of NF-κB p65 in the process of arsenic(AS)-induced apoptosis of liver cancer cells.Methods:Human hepatoma cells(HepG2 cells)were cultured in vitro,and lentiviral transfection was used to construct a HepG2 cell line that overexpressed/knocked down QRICH1 stably.They were divided into control group,arsenic addition group,arsenic^(+)QRICH1 overexpression negative control group,arsenic^(+)QRICH1 overexpression group,arsenic^(+)QRICH1knockdown negative control group and arsenic^(+)QRICH1 knockdown group.In the logarithmic growth phase of cells,40μmol/L sodium arsenite(NaAsO2)was added as the final concentration for 24 h.The control group was treated with the same volume of phosphate buffered saline(PBS)as NaAsO2.Cell proliferation was detected by cell counting(CCK-8).Cell apoptosis in each group was detected by flow cytometry.Western blot was used to detect the expression levels of QRICH1,NF-κB p65,Bcl-2 and Bax proteins in each group of cells.Results:The results of CCK-8showed that compared with the control group,the proliferation rate of the arsenic treatment group was significantly lower(P<0.05).Compared with the arsenic^(+)QRICH1 overexpression negative control group,the proliferation rate of the arsenic^(+)QRICH1 overexpression group was significantly increased(P<0.05).Compared with the arsenic^(+)QRICH1 knockdown negative control group,the cell proliferation rate in the arsenic^(+)QRICH1 knockdown group was significantly decreased(P<0.05),and the difference was statistically significant.The results of flow cytometry showed that compared with the control group,the proportion of total apoptosis in the arsenic treatment group was significantly increased(P<0.05).Compared with the arsenic^(+)QRICH1 overexpression negative control group,the total apoptosis rate of the arsenic^(+)QRICH1 overexpression group was significantly decreased.Compared with the arsenic^(+)QRICH1 knockdown negative control group,the proportion of total apoptosis rate in the arsenic^(+)QRICH1 knockdown group was significantly increased(P<0.05).Western blotting results showed that compared with the control group,the expression levels of QRICH1,NF-κB p65 and Bcl-2 proteins in the arsenic treatment group were lower,and the protein expression level of Bax was higher(P<0.05).Compared with the arsenic^(+)QRICH1 overexpression negative control group,the arsenic^(+)QRICH1 overexpression group had higher protein expression levels of QRICH1,NF-κB p65 and Bcl-2,and lower protein expression levels of Bax(P<0.05).Compared with the arsenic^(+)QRICH1 knockdown negative control group,the arsenic^(+)QRICH1 knockdown group had lower protein expression levels of QRICH1,NF-κB p65 and Bcl-2,and higher protein expression levels of Bax(P<0.05),and the difference was statistically significant.Conclusion:In the process of arsenic-induced apoptosis of HepG2,QRICH1 affects the expression of Bcl-2 and Bax by regulating the expression of NF-κB p65.It is suggested that QRICH1 may be a potential target for promoting apoptosis of liver cancer cells.