敲减CCAAT/增强子结合蛋白α通过增加O-GlcNAc糖基化水平促进肝癌细胞体外增殖

摘要

目的 探讨CCAAT/增强子结合蛋白α(C/EBPα)在肝癌细胞增殖中的作用及其机制.方法 利用靶向C/EBPα的RNAi慢病毒感染Hep3B肝癌细胞系,构建C/EBPα肝癌细胞系敲减模型.采用 qRT-PCR和Western blot分别检测C/EBPα mRNA和蛋白表达水平,采用CCK-8检测敲减C/EBPα后对Hep3B细胞增殖的影响,采用在正常高葡萄糖(NG,4.5 g/L)和低葡萄糖(LG,1 g/L)条件下培养细胞的增殖变化,采用Western blot法检测不同葡萄糖浓度条件下在C/EBPα敲减细胞和对照细胞乙酰葡糖胺转移酶(OGT)、乙酰葡糖胺水解酶(OGA)和整个O-GlcNAc糖基化水平.结果 靶向C/EBPα的RNAi慢病毒感染Hep3B肝癌细胞后,C/EBPα mRNA水平下调了(7.5±2.3)倍(P<0.05),蛋白表达水平下调了(8.8±0.25)倍(P<0.001),提示C/EBPα敲减细胞系构建成功;敲减C/EBPα后明显促进肝癌细胞的体外增殖;在低葡萄糖条件下刺激48 h和72 h,敲减C/EBPα分别促进细胞增殖15.4%(P<0.05)和25.0%(P<0.01);敲减C/EBPα后细胞OGA蛋白表达水平在正常高糖和低糖刺激24 h和48 h时分别下调了70.1%(P<0.01)、51.4%(P<0.05)和61.2%(P<0.05),整体O-GlcNAc糖基化表达水平上调,在低糖刺激48 h时升高80.6%.结论 敲减转录因子C/EBPα可以提高细胞整体O-GlcNAc糖基化水平,从而促进肝癌细胞的体外增殖.%Objective To explore the roles and potential mechanism of CCAAT/enhancer-binding protein alpha(C/EBPα)in the growth of hepatocellular carcinoma(HCC)cells in vitro. Methods The Hep3B cells were transfected with RNAi lentivirous,and the mRNA and protein of C/EBPα were detected by qRT-PCR and Western blot respectively. The growth of HCC cells in normal glucose(NG,4.5 g/L)and low glucose(LG,1 g/L) were assessed by CCK-8 assay and quantified based on the absorbance value at OD450. The protein levels of O-GlcNAcase(OGA),O-GlcNAc transferase(OGT)and the whole O-GlcNAcylation in normal glucose and low glucose were detected by Western blot. Results The C/EBPαmRNA level was down-regulated by(7.5±2.3)folds (P<0.05)and its protein was decreased by(8.8±0.25)folds(P<0.001)in transfected with RNAi lentivirous cells (sh-C/EBPα)compared with control cell(sh-Con). Knocking down C/EBPα promoted the growth of HCC cells in vitro. The growth rates of sh-C/EBPα cells increased by 15.4%(P<0.05)and 25.0%(P<0.01)in low glucose (LG,1 g/L)condition at 48 hours and 72 hours;The protein of OGA decreased by 70.1%(P<0.01),51.4%(P<0.05)and 6 1.2%(P<0.05)in normal gulucose,low glucose at 24 h and 48 h. Whole O-GlcNAcylation was up-regulated by 80.6% in the C/EBPα knocked-down cells in low glucose at 48 h. Conclusions The downregulation of CCAAT/enhancer-binding protein alpha promotes the growth of hepatocellular carcinoma cells in vitro likely by increasing O-GlcNAcylation in some extent.