HBX在肝癌细胞中通过下调miR-199a增强AKT的表达

HBX enhances AKT expression through down-regulation of miR-199a in hepatitis B virus related hepatocellular carcinoma

目的探讨乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBX)调节miR-199a的表达在HBV相关性肝癌发生中的分子机制。方法重组HBX腺病毒(Ad-HBX)感染人肝癌Hep G2细胞,HBX的siRNA转染稳定表达HBV基因的人肝癌Hep G2.2.15细胞,荧光定量PCR方法检测HBV相关性肝癌组织标本和肝癌细胞中HBX、miR-199a、AKT的mRNA表达水平及运用Western blot检测肝癌组织标本和肝癌细胞中HBX、AKT的蛋白表达水平;分别用miR-199a mimics和miR-199a inhibitor的转染肝癌细胞后,荧光定量PCR方法检测肝癌细胞中AKT的mRNA表达水平及运用Western blot检测肝癌细胞中AKT的蛋白表达水平。结果 miR-199a在Hep G2.2.15细胞中表达水平显著低于Hep G2细胞,并且证实其表达下调受到了HBX的调控(P<0.05),Hep G2.2.15细胞中AKT表达水平显著高于Hep G2细胞(P<0.05),在Hep G2.2.15细胞中过表达miR-199a能明显下调AKT表达水平以及在Hep G2细胞中抑制miR-199a能明显上调的AKT表达水平(P<0.05),此外,miR-199a的表达量在HBV相关性肝癌组织比相应的癌旁组织表达降低。结论 HBX可以通过miR-199a调节AKT导致HBV相关性肝癌发生。

Objective To explore the potential mechanism of hepatitis B virus X protein （HBX） - modulated miR-199a expression in hepatitis B virus （HBV） related hepatocarcinogenesis. Methods HepG2 cells were transfected with HBX-containing recombinant adenovirus （Ad-HBX）, and HepG2. 2. 15 cells capable of stably expressing HBV gene were transfected with siRNA-HBX. Fluorescence quantitative PCR was used for detecting mRNA expression levels of HBX, miR-199a, and AKT in HBV-related hepatocellular carcinoma tissues and hepatocellular carcinoma cells, while Western blotting was adopted to detect the protein expression of HBX and AKT. After transfection with miR-199a mimics and miR-199a inhibitor, quantitative PCR and Western blotting were employed to detect the expression of AKT in hepatocellular carcinoma ceils. Results Experimental results showed that miR-199a expression in HepG2.2.15 cells was significantly lower than that in HepG2 cells, and down-regulation of miR-199a expression was related with HBX （ P 〈 0. 05 ）. AKT expression in HepG2.2.15 cells was significantly higher than that in HepG2 ceils （P 〈 0. 05 ）, and over- expression of miR-199a in HepG2. 2.15 cells could significantly reduce AKT expression （ P 〈 0. 05 ）, while inhibition of miR-199a in HepG2 cells could significantly increase AKT expression （P 〈0.05 ）. In addition, the expression level of miR-199a in HBV-related hepatocellular carcinoma tissues was lower than that in the adjacent noncancer tissues. Conclusion HBX regulates the AKT expression through miR-199a, which may lead to HBV-related hepatocarcinogenesis.