Original Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Oct 7, 2013; 19(37): 6178-6187
Published online Oct 7, 2013. doi: 10.3748/wjg.v19.i37.6178
Cytokeratin 8 is increased in hepatitis C virus cells and its ectopic expression induces apoptosis of SMMC7721 cells
Ming-Zhu Sun, Shuang-Suo Dang, Wen-Jun Wang, Xiao-Li Jia, Song Zhai, Xin Zhang, Mei Li, Ya-Ping Li, Meng Xun
Ming-Zhu Sun, Shuang-Suo Dang, Wen-Jun Wang, Xiao-Li Jia, Song Zhai, Xin Zhang, Mei Li, Ya-Ping Li, Department of Infectious Diseases, the Second Affiliated Hospital of College of Medicine, Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China
Ming-Zhu Sun, Shuang-Suo Dang, Wen-Jun Wang, Xiao-Li Jia, Song Zhai, Xin Zhang, Mei Li, Ya-Ping Li, Department of Epidemiology and Biostatistics, College of Medicine, Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China
Meng Xun, Department of Immunology and Pathogen Biology, College of Medicine, Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China
Author contributions: Sun MZ wrote the manuscript; Dang SS supervised the research process and revised the manuscript; Wang WJ critically revised the manuscript; Jia XL, Zhai S, Zhang X, Li M, Li YP and Xun M collected data; all authors have read and approved the final version to be published.
Supported by National Natural Science Foundation of China, No.81170393
Correspondence to: Shuang-Suo Dang, PhD, Professor, Director of Department of Infectious Diseases, the Second Affiliated Hospital of College of Medicine, Xi’an Jiaotong University, No. 157, Xi Wu Road, Xi’an 710004, Shaanxi Province, China. dang212@126.com
Telephone: +86-29-87679688 Fax: +86-29-87679688
Received: June 30, 2013
Revised: August 10, 2013
Accepted: August 16, 2013
Published online: October 7, 2013
Abstract

AIM: To investigate cytokeratin 8 (CK8) overexpression during hepatitis C virus (HCV) infection and its pathogenesis, and the effect of ectopic CK8 expression on hepatoma cell lines.

METHODS: We successfully established an in vitro HCV cell culture system (HCVcc) to investigate the different expression profiles of CK8 in Huh-7-HCV and Huh-7.5-HCV cells. The expression of CK8 at the mRNA level was determined by real-time polymerase chain reaction (RT-PCR). The expression of CK8 at the protein level was evaluated by Western blotting. We then constructed a eukaryotic expression combination vector containing the coding sequence of human full length CK8 gene. CK8 cDNA was amplified by reverse transcription-PCR and inserted into pEGFP-C1 and the positive clone pEGFP-CK8 was obtained. After confirming the sequence, the recombinant plasmid was transfected into SMMC7721 cells with lipofectamine2000 and CK8 expression was detected using inverted fluorescence microscopy, RT-PCR and Western blotting. Besides, we identified biological function of CK8 on SMMC7721 cells, including cell proliferation, cell cycle and apoptosis detection.

RESULTS: RT-PCR showed that the expression level of CK8 in Huh-7-HCV and Huh-7.5-HCV cells was 2.88 and 2.95 times higher than in control cells. Western blot showed that CK8 expression in Huh-7-HCV and Huh-7.5-HCV cells was 2.53 and 3.26 times higher than that in control cells, respectively. We found that CK8 at mRNA and protein levels were both significantly increased in HCVcc. CK8 was up-regulated in SMMC7721 cells. CK8 expression at the mRNA level was significantly upregulated in SMMC7721/pEGFP-CK8 cells. CK8 expression in SMMC7721/ pEGFP-CK8 cells was 2.69 times higher than in SMMC7721 cells, and was 2.64 times higher than in SMMC7721/pEGFP-C1 cells. CK8 expression at the protein level in SMMC7721/pEGFP-CK8 cells was 2.46 times higher than in SMMC7721 cells, and was 2.29 times higher than in SMMC7721/pEGFP-C1 cells. Further analysis demonstrated that forced expression of CK8 slowed cell growth and induced apoptosis of SMMC7721 cells.

CONCLUSION: CK8 up-regulation might have a functional role in HCV infection and pathogenesis, and could be a promising target for the treatment of HCV infection.

Keywords: Hepatitis C virus cell culture system, Cytokeratin 8, Up-regulation, Eukaryotic expression, Apoptosis

Core tip: In this study, we observed that cytokeratin 8 (CK8) levels are elevated in hepatitis C virus (HCV) cell culture system and its ectopic expression decreased the proliferation and induced apoptosis of SMMC7721 cells. CK8 up-regulation might have a functional role in HCV infection and pathogenesis, and could be a promising target for the treatment of HCV infection.