Hepatitis C Virus Nonstructural 5A Protein (HCV-NS5A) Inhibits Hepatocyte Apoptosis through the NF-κb/miR-503/bcl-2 Pathway.
The nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus (HCV) RNA genome is a multifunctional phosphoprotein. To analyse the influence of NS5A on apoptosis, we established an Hep-NS5A cell line (HepG2 cells that stably express NS5A) and induced apoptosis using tumour necrosis factor (TNF)-α. We utilised the MTT assay to detect cell viability, real-time quantitative polymerase chain reaction and Western blot to analyse gene and protein expression, and a luciferase reporter gene experiment to investigate the targeted regulatory relationship. Chromatin immunoprecipitation was used to identify the combination of NF-κB and miR-503. We found that overexpression of NS5A inhibited TNF-αinduced hepatocellular apoptosis via regulating miR-503 expression. The cell viability of the TNF-α induced Hep-mock cells was significantly less than the viability of the TNF-α induced Hep-NS5A cells, which demonstrates that NS5A inhibited TNF-α-induced HepG2 cell apoptosis. Under TNF-α treatment, miR-503 expression was decreased and cell viability and B-cell lymphoma 2 (bcl-2) expression were increased in the Hep-NS5A cells. Moreover, the luciferase reporter gene experiment verified that bcl-2 was a direct target of miR-503, NS5A inhibited TNF-α-induced NF-κB activation and NF-κB regulated miR-503 transcription by combining with the miR-503 promoter. After the Hep-NS5A cells were transfected with miR-503 mimics, the data indicated that the mimics could reverse TNF-α-induced cell apoptosis and blc-2 expression. Collectively, our findings suggest a possible molecular mechanism that may contribute to HCV treatment in which NS5A inhibits NF-κB activation to decrease miR-503 expression and increase bcl-2 expression, which leads to a decrease in hepatocellular apoptosis.
Department of Gastroenterology, The Second Affiliated Hospital, Nanchang University, Nanchang 330006, China.
Zhengyuan
Z
Xie
Department of Gastroenterology, The Second Affiliated Hospital, Nanchang University, Nanchang 330006, China.
Zhihua
Z
Xiao
Department of Gastroenterology, The Second Affiliated Hospital, Nanchang University, Nanchang 330006, China.
Fenfen
F
Wang
D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival; D005786:Gene Expression Regulation; D016174:Hepacivirus / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D035683:MicroRNAs / Q000235:genetics; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
D035683:MicroRNAs; D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D017361:Viral Nonstructural Proteins; C545473:MIRN503 microRNA, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
HCV-NS5A; NF-κB; bcl-2; hepatocyte apoptosis; miR-503