Comparative analysis of gene expression upon IFNa stimulation in hepatocyte-like cells [IFN]

Patients chronically co-infected by Hepatitis B Virus (HBV) and Hepatitis D Virus (HDV) suffer from the most aggressive form of viral hepatitis leading to severe liver disease such as cirrhosis, hepatocellular carcinoma and liver decompensation. Treatments options are very limited with only two drugs used in clinic (IFN-a and Bulevertide) that rarely allow viral clearance. The molecular mechanisms leading to inhibition of HDV by IFN-a (in patients but also in vitro) are not known and mechanisms behind treatment failures in patients also remain elusive. Here we aimed at the identification of interferon stimulated genes (ISGs) that can specifically inhibit intracellular HDV replication. IFN-a was able to inhibit HDV in both co-infected differentiated HuH7.5-NTCP- and HepaRG cells (dHuH7.5-NTCP and dHepaRG, respectively). We hypothesized that ISGs responsible for the antiviral effect of HDV should be upregulated in both dHepaRG and dHuH7.5-NTCP cells upon treatment with IFN-a. To identify these ISGs, dHuh7.5-NTCP and dHepaRG cells were stimulated with IFN-a for 8h, total RNAs were extracted and gene expression was analyzed by RNAseq. Overall design: differentiated HepaRG cells and differentiated HuH7.5-NTCP cells were stimulated or not with IFNa-2a (500 UI/mL) for 8h. Total RNAs were extracted and gene expression was analyzed by RNAseq

慢性乙型肝炎病毒（Hepatitis B Virus, HBV）与丁型肝炎病毒（Hepatitis D Virus, HDV）共感染的患者，会罹患病情最为凶险的病毒性肝炎亚型，进而引发肝硬化、肝细胞癌、肝失代偿等严重肝脏疾病。当前临床可用治疗方案极为有限，仅获批两款药物（干扰素α（IFN-α）与布列韦肽），且二者极少能实现病毒学清除。干扰素α抑制HDV的分子机制——无论是在患者体内还是体外实验体系中——均未阐明，而患者治疗失败的背后机制也仍不明晰。本研究旨在筛选可特异性抑制细胞内HDV复制的干扰素刺激基因（Interferon Stimulated Genes, ISGs）。实验证实，干扰素α可在共感染的分化型HuH7.5-NTCP细胞与HepaRG细胞（分别记为dHuH7.5-NTCP与dHepaRG）中有效抑制HDV复制。据此我们推测，介导干扰素α抗HDV效应的干扰素刺激基因，应在经干扰素α处理后的dHepaRG与dHuH7.5-NTCP细胞中呈现上调表达。为筛选得到此类干扰素刺激基因，我们将dHuH7.5-NTCP与dHepaRG细胞用干扰素α处理8小时，随后提取总RNA，通过RNA测序（RNAseq）分析基因表达情况。整体实验设计：分别对分化型HepaRG细胞与分化型HuH7.5-NTCP细胞施加或不施加500 UI/mL的干扰素α-2a处理，持续8小时。之后提取总RNA，通过RNA测序分析基因表达水平。