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*

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。

Downregulation of CCAAT/enhancer-binding protein alpha promotes growth of hepatocellular carcinoma cells in vitro by increasing O-GlcNAcylation

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. Conclusion s 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.