缺失突变型HBx蛋白介导PDK2在肝细胞肝癌中的差异表达及机制

Differential expression and mechanism of PDK2 mediated by deletion mutant HBx protein in hepatocellular carcinoma

目的探讨肝细胞肝癌(HCC)中自然缺失突变型乙型肝炎病毒X(HBx)蛋白介导的丙酮酸脱氢酶激酶2(PDK2)的差异表达情况以及可能参与调控长链非编码RNA(lncRNA)的机制。方法将Huh7细胞随机分为空载体pcDNA3.1组和HBx-128w组,分别转染pcDNA3.1和pc DNA3.1-HBx-128w。采用Western blotting法检测HBx蛋白的表达。使用lncRNA芯片对两组稳转细胞进行检测,明确pc DNA3.1-HBx-128w转染的Huh7细胞的m RNA表达谱和lncRNA表达谱,筛选相关差异表达基因,利用RT-qPCR方法在稳转细胞中进行验证。利用生物信息学预测可能参与调控的lncRNA,并利用RT-q PCR方法在稳转细胞中进行验证。结果PDK2在HBx-128w组Huh7细胞中呈上调表达,Fold Change为3.219。HBx-128w组Huh7细胞中PDK2的浓度比pcDNA3.1组升高(P<0.05),两组间差异倍数为3.591。HBx-128w组中的lncRNA ENST0000511361表达量较pc DNA3.1组升高(P<0.05),两组间差异倍数为3.742。结论自然缺失突变型HBx-128w转染的Huh7细胞中,PDK2呈上调表达;LncRNA ENST0000511361可能参与HCC发展过程中PDK2表达的调控。

Objective To investigate the differential expression of PDK2 mediated by natural deletion mutant hepatitis B virus X(HBx)protein in hepatocellular carcinoma(HCC),as well as the mechanism of long non-coding RNA(lncRNA)involved in regulation.Methods Huh7 cells were randomly divided into the empty vector group and HBx-128 w group,and transfected with pcDNA3.1 and pcDNA3.1-HBx-128 w,respectively.Western blotting was used to detect the expression of HBx protein in each group.LncRNA microarray was used to detect the stable transfected cells in the two groups,and the mRNA expression profiles and lncRNA expression profiles of Huh7 cells transfected with pcDNA3.1-HBx-128 w were determined,and the differentially expressed genes were screened out.RT-qPCR was used to verify the results of lncRNA microarray in stable transfected cells.Bioinformatics was used to predict IncRNAs that might be involved in regulation,and RT-qPCR was used for verification in stable transfected cells.Results The lncRNA microarray results showed that PDK2 was up-regulated in Huh7 cells of HBx-128 w group,and the fold change was 3.219.RT-qPCR results showed that the concentration of PDK2 in Huh7 cells in the HBx-128 w group was significantly higher than that in the pcDNA3.1 group(P<0.05),and the fold change between the two groups was 3.591.The expression level of lncRNA ENST0000511361 in HBx-128 w group was significantly higher than that in the pcDNA3.1 group(P<0.05),and the fold change between the two groups was 3.742.Conclusion PDK2 is up-regulated in Huh7 cells transfected with natural deletion mutant HBx-128 w,and LncRNA ENST0000511361 may be involved in the regulation of PDK2 expression in the development of HCC.