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q RT-PCR和Western blot分别检测C/EBPαm RNA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-Glc NAc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/EBPαm RNA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-Glc NAc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-Glc NAc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 proteinalpha （C/EBPα）in the growth of hepatocellular carcinoma （HCC）cells in vitro. Methods The Hep3B cellswere transfected with RNAi lentivirous, and the mRNA and protein of C/EBPα were detected by qRT-PCR andWestern 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 lowglucose 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 invitro. 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 downregulationof CCAAT/enhancer-binding protein alpha promotes the growth of hepatocellular carcinoma cells in vitro likely byincreasing O-GlcNAcylation in some extent.
作者
李文姣
曹海超
马澜婧
张松
丁美玲
张浩浩
聂勇战
Li Wenjiao;Cao Haichao;Ma Lanjing(StateKey Laboratory of Cancer Biology, Xijing Hospital of Digestive Disease, Affiliated to Fourth Military MedicalUniversity, Xi＇an, 710032, China)
出处
《实用肝脏病杂志》
CAS
2018年第3期393-396,共4页
Journal of Practical Hepatology
基金
国家杰出青年科研基金资助项目(编号:81225003)
