National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
Vice Chancellor Office, Hazara University, Mansehra, Pakistan.
J Med Virol. 2018 Sep;90(9):1516-1523. doi: 10.1002/jmv.25221. Epub 2018 Jun 11.
Epigenetic modifications such as DNA methylation contribute to progression of hepatitis C virus (HCV) infection to life-threatening hepatocellular carcinoma (HCC) by promoting the silencing of tumor suppressor genes through DNA hypermethylation and by causing genomic instability through global hypomethylation. However few studies have addressed the promoter region hypomethylation status of the oncogenes involved in HCV derived HCC. In this study, we analyzed the promoter region methylation pattern of RAS oncogenes (HRAS, KRAS, and NRAS) using methylation-specific PCR for 50 chronic HCV patients infected with genotype 3a (27 HCC patients and 23 control non-HCC patients). Methylation-specific polymerase chain reaction analysis revealed that the NRAS oncogene promoter (P = .0025) was significantly hypomethylated in HCC patients compared to the non-HCC patients suggesting its contribution to the progression of HCV towards HCC. To identify the agent for alteration in the RAS oncogene expression, 7 HCV genes were expressed in the Huh-7 cell line followed by measurement of the NRAS expression level in Huh-7 by a quantitative real-time polymerase chain reaction. An increase in the messenger RNA level of the NRAS gene was detected when Huh-7 were transfected with Core, NS5a, and NS2 genes. Our findings suggest the involvement of NRAS oncogene in the pathogenesis of HCV3a derived HCC in Pakistani population and also identifies the HCV genes responsible for its enhanced expression. Our study raises the hypothesis that a single HCV gene may increase the chances of malignancy. Therefore, our study may have identified a useful epigenetic biomarker of HCC progression in HCV patients and may help to develop novel diagnostic tools.
表观遗传修饰，如 DNA 甲基化，通过 DNA 超甲基化促进肿瘤抑制基因的沉默，并通过全基因组低甲基化引起基因组不稳定性，从而促进丙型肝炎病毒 (HCV) 感染向致命性肝细胞癌 (HCC) 的进展。然而，很少有研究涉及与 HCV 衍生 HCC 相关的癌基因启动子区低甲基化状态。在这项研究中，我们使用甲基化特异性 PCR 分析了 50 例感染基因型 3a 的慢性 HCV 患者（27 例 HCC 患者和 23 例非 HCC 对照患者）的 RAS 癌基因（HRAS、KRAS 和 NRAS）启动子区甲基化模式。甲基化特异性聚合酶链反应分析显示，与非 HCC 患者相比，HCC 患者 NRAS 癌基因启动子（P=0.0025）明显低甲基化，提示其对 HCV 向 HCC 进展的贡献。为了确定 RAS 癌基因表达改变的原因，我们在 Huh-7 细胞系中表达了 7 种 HCV 基因，然后通过定量实时聚合酶链反应测量 Huh-7 中的 NRAS 表达水平。当 Huh-7 转染 Core、NS5a 和 NS2 基因时，NRAS 基因的信使 RNA 水平增加。我们的研究结果表明，NRAS 癌基因参与了巴基斯坦人群 HCV3a 衍生 HCC 的发病机制，并确定了导致其表达增强的 HCV 基因。我们的研究提出了一个假设，即单个 HCV 基因可能会增加恶性肿瘤的机会。因此，我们的研究可能已经确定了 HCV 患者 HCC 进展的有用表观遗传生物标志物，并可能有助于开发新的诊断工具。
