Supplementary Material for: Identification and Verification of Ubiquitin D as a gene associated with HCV-induced HCC

Introduction: Accumulated studies have suggested that hepatitis C virus (HCV) infection is one of the leading causes for hepatocellular carcinoma (HCC). However, the mechanisms underlying the effect of HCV on the occurrence of HCC is still poorly understood. Methods: HCV infection datasets (GSE82177 and GSE17856) and HCC datasets (TCGA-LIHC (The Cancer Genome Atlas Liver Hepatocellular Carcinoma) and GSE89377) were downloaded from Gene Expression Omnibus (GEO) or TCGA for analysis. The common differentially expressed genes (DEGs) in the above four datasets were identified by R software. The expression of Ubiquitin D (UBD) in HCV infected HepG2 cells was detected by RT-qPCR and western blot, respectively. The interaction between NS3 and p53 was detected by Co-Immunoprecipitation. The influence of UBD on the proliferation and migration ability of HepG2 cells was evaluated by CCK-8 and wound healing assay, respectively. Results: UBD was upregulated in both HCV infected samples and HCC samples. HCV NS3 interacted with p53 and inhibited its expression. HCV NS3 induced UBD promoted the proliferation and migration of HepG2 cells. Conclusion: Our results suggest that HCV NS3 induced UBD is positively correlated with the development of HCV-related HCC during HCV infection. Targeting UBD could be a potential strategy for preventing and treating HCV-induced HCC.

引言：已有多项研究表明，丙型肝炎病毒（hepatitis C virus, HCV）感染是肝细胞癌（hepatocellular carcinoma, HCC）的主要致病诱因之一。然而，HCV诱发肝细胞癌发生的具体分子机制目前仍不甚明确。
研究方法：本研究从基因表达综合数据库（Gene Expression Omnibus, GEO）与癌症基因组图谱（The Cancer Genome Atlas, TCGA）中下载HCV感染数据集（GSE82177、GSE17856）及肝细胞癌数据集（TCGA-LIHC（The Cancer Genome Atlas Liver Hepatocellular Carcinoma）、GSE89377）用于后续分析。通过R软件筛选出上述4组数据集中共有的差异表达基因（differentially expressed genes, DEGs）。分别采用实时荧光定量聚合酶链式反应（RT-qPCR）与蛋白质印迹法（western blot）检测HCV感染的HepG2细胞中泛素D（Ubiquitin D, UBD）的表达水平；通过免疫共沉淀（Co-Immunoprecipitation）验证NS3蛋白与p53蛋白的相互作用；分别采用细胞计数试剂盒-8（CCK-8）与划痕愈合实验（wound healing assay）评估UBD对HepG2细胞增殖与迁移能力的影响。
研究结果：UBD在HCV感染样本与肝细胞癌样本中均呈现上调表达。HCV NS3蛋白可与p53蛋白结合并抑制其表达。HCV NS3诱导的UBD可促进HepG2细胞的增殖与迁移能力。
研究结论：本研究结果表明，在HCV感染过程中，HCV NS3蛋白诱导的UBD上调表达与HCV相关性肝细胞癌的发生发展呈正相关。靶向干预UBD或可成为预防及治疗HCV诱发肝细胞癌的潜在策略。