Hepatitis C Virus Nonstructural 5A Protein (HCV-NS5A) Inhibits Hepatocyte Apoptosis through the NF-κb/miR-503/bcl-2 Pathway

  • Xie, Zhengyuan (Department of Gastroenterology, The Second Affiliated Hospital, Nanchang University) ;
  • Xiao, Zhihua (Department of Gastroenterology, The Second Affiliated Hospital, Nanchang University) ;
  • Wang, Fenfen (Department of Gastroenterology, The Second Affiliated Hospital, Nanchang University)
  • Received : 2016.12.07
  • Accepted : 2017.02.22
  • Published : 2017.03.31


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 $(TNF)-{\alpha}$. 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 $NF-{\kappa}B$ and miR-503. We found that overexpression of NS5A inhibited $TNF-{\alpha}$-induced hepatocellular apoptosis via regulating miR-503 expression. The cell viability of the $TNF-{\alpha}$ induced Hep-mock cells was significantly less than the viability of the $TNF-{\alpha}$ induced Hep-NS5A cells, which demonstrates that NS5A inhibited $TNF-{\alpha}$-induced HepG2 cell apoptosis. Under $TNF-{\alpha}$ 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 $TNF{\alpha}$-induced $NF-{\kappa}B$ activation and $NF-{\kappa}B$ 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 $TNF-{\alpha}$-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 $NF-{\kappa}B$ activation to decrease miR-503 expression and increase bcl-2 expression, which leads to a decrease in hepatocellular apoptosis.


bcl-2;HCV-NS5A;hepatocyte apoptosis;miR-503;$NF-{\kappa}B$


Supported by : National Natural Science Foundation of China, The Second Affiliated Hospital of Nanchang University


  1. Bandiera, S., Pernot, S., El Saghir, H., Durand, S.C., Thumann, C., Crouchet, E., Ye, T., Fofana, I., Oudot, M.A., Barths, J., et al. (2016). Hepatitis C virus-induced upregulation of microRNA miR-146a-5p in hepatocytes promotes viral infection and deregulates metabolic pathways associated with liver disease pathogenesis. J. Virol. 90, 6387-6400.
  2. Bartel, D.P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281-297.
  3. Caporali, A., Meloni, M., Nailor, A., Mitic, T., Shantikumar, S., Riu, F., Sala-Newby, G.B., Rose, L., Besnier, M., Katare, R., et al. (2015). $P75^{NTR}$-dependent activation of $NF-{\kappa}B$ regulates microRNA-503 transcription and pericyte-endothelial crosstalk in diabetes after limb ischaemia. Nat. Commun. 6, 8024.
  4. Charlton, M., Everson, G.T., Flamm, S.L., Kumar, P., Landis, C., Jr, B.R., Fried, M.W., Terrault, N.A., O'Leary, J.G., Vargas, H.E., et al. (2015). Ledipasvir and Sofosbuvir Plus Ribavirin for Treatment of HCV Infection in Patients With Advanced Liver Disease. Gastroenterology 149, 649-659.
  5. Chen, X., Hu, H., Guan, X., Xiong, G., Wang, Y., Wang, K., Li, J., Xu, X., Yang, K., and Bai, Y. (2012). CpG island methylation status of miRNAs in esophageal squamous cell carcinoma. Int. J. Cancer 130, 1607-1613.
  6. Coburn, C.A., Meinke, P.T., Chang, W., Fandozzi, C.M., Graham, D.J., Hu, B., Huang, Q., Kargman, S., Kozlowski, J., Liu, R., et al. (2013). Discovery of MK-8742: an HCV NS5A inhibitor with broad genotype activity. ChemMedChem 8, 1930-1940.
  7. Gane, E., Poordad, F., Wang, S., Asatryan, A., Kwo, P.Y., Lalezari, J., Wyles, D.L., Hassanein, T., Aguilar, H., Maliakkal, B., et al. (2016). High efficacy of ABT-493 and ABT-530 treatment in patients with HCV genotype 1 or 3 infection and compensated cirrhosis. Gastroenterology 151, 651-659.e1.
  8. Gao, M., Nettles, R.E., Belema, M., Snyder, L.B., Nguyen, V.N., Fridell, R.A., Serrano-Wu, M.H., Langley, D.R., Sun, J.H., O'Boyle, D.R., 2nd., et al. (2010). Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 465, 96-100.
  9. Ikeda, H., Watanabe, T., Okuse, C., Matsumoto, N., Ishii, T., Yamada, N., Shigefuku, R., Hattori, N., Matsunaga, K., and Nakano, H. (2016). Impact of resistance-associated variant dominancy on treatment in patients with HCV genotype 1b receiving daclatasvir/asunaprevir. Journal of Medical Virology 89, 99-105.
  10. Ishida, H., Tatsumi, T., Hosui, A., Nawa, T., Kodama, T., Shimizu, S., Hikita, H., Hiramatsu, N., Kanto, T., and Hayashi, N. (2011). Alterations in microRNA expression profile in HCV-infected hepatoma cells: Involvement of miR-491 in regulation of HCV replication via the PI3 kinase/Akt pathway. Biochem. Biophys. Res. Commun. 412, 92-97.
  11. Lawitz, E., Poordad, F.F., Pang, P.S., Hyland, R.H., Ding, X., Mo, H., Symonds, W.T., Mchutchison, J.G., and Membreno, F.E. (2014). Sofosbuvir and ledipasvir fixed-dose combination with and without ribavirin in treatment-naive and previously treated patients with genotype 1 hepatitis C virus infection (LONESTAR): an open-label, randomised, phase 2 trial. Lancet 383, 515-523.
  12. Li, S., Duan, X., Li, Y., Liu, B., Mcgilvray, I., and Chen, L. (2014). MicroRNA-130a inhibits HCV replication by restoring the innate immune response. J. Viral Hepat. 21, 121-128.
  13. Lim, E.J., El, K.K., Chin, R., Earnest-Silveira, L., Angus, P.W., Bock, C.T., Nachbur, U., Silke, J., and Torresi, J. (2014). Hepatitis C virusinduced hepatocyte cell death and protection by inhibition of apoptosis. J. Gen. Virol. 95, 2204-2215.
  14. Lin, C.W., Jones, A.K., Liu, W., and Dutta, S. (2015). Population pharmacokinetics of a pangenetic NS5A inhibitor, ABT-530, in HCV infected patients with and without cirrhosis. J. Pharmacokinet. Pharmacodyn. 42, S86.
  15. Ling, T., Yu, W., Lei, C., Selyutin, O., Dwyer, M.P., Nair, A.G., Mazzola, R., Kim, J.H., Sha, D., Yin, J., et al. (2016). The discovery of ruzasvir (MK-8408): a potent, pan-genotype HCV NS5A inhibitor with optimized activity against common resistance-associated polymorphisms. J. Med. Chem. 60, 290-306.
  16. Liu, Y.P., Lee, J.J., Lai, T.C., Lee, C.H., Hsiao, Y.W., Chen, P.S., Liu, W.T., Hong, C.Y., Lin, S.K., Ping, K.M., e t al. (2015). Suppressive function of low-dose deguelin on the invasion of oral cancer cells by down-regulating TNF-${\alpha}$-induced $NF-{\kappa}B$ signaling. Head Neck 38, E524-E534.
  17. Majumder, M., Ghosh, A.K., Steele, R., Zhou, X.Y., Phillips, N.J., Ray, R., and Ray, R.B. (2002a). Hepatitis C virus NS5A protein impairs TNFmediated hepatic apoptosis, but not by an anti-FAS antibody, in transgenic mice. Virology 294, 94-105.
  18. Majumder, M., Ghosh, A.K., Steele, R., Zhou, X.Y., Phillips, N.J., Ray, R., and Ray, R.B. (2002b). Hepatitis C virus NS5A protein impairs TNFmediated hepatic apoptosis, but not by an anti-FAS antibody, in transgenic mice. Virology 294, 94-105.
  19. Mitupatum, T., Aree, K., Kittisenachai, S., Roytrakul, S., Puthong, S., Kangsadalampai, S., and Rojpibulstit, P. (2016). mRNA expression of Bax, Bcl-2, p53, Cathepsin B, Caspase-3 and Caspase-9 in the HepG2 cell line following induction by a novel monoclonal Ab Hep88 mAb: cross-talk for paraptosis and apoptosis. Asian Pac. J. Cancer Prev. 17, 703-712.
  20. Miyasaka, Y., and Watanabe, M. (2003). Hepatitis C virus nonstructural protein 5A inhibits tumor necrosis factor-alphamediated apoptosis in Huh7 cells. J. Infect. Dis. 188, 1537-1544.
  21. Mogalian, E., Mathias, A., Brainard, D., Shen, G., Mcnally, J., Sajwani, K., Robson, R., Ries, D., Lasseter, K., and Marbury, T. (2015). P0712 : The pharmacokinetics of GS-5816, a pangenotypic HCV-specific NS5A inhibitor, in HCV-uninfected subjects with severe renal impairment. J. Hepatol. 62, S590-S591.
  22. Organization, W.H. (2015). EN fact sheets: hepatitis C.
  23. Qiu, T., Zhou, L., Wang, T., Xu, J., Wang, J., Chen, W., Zhou, X., Huang, Z., Zhu, W., and Shu, Y. (2013). miR-503 regulates the resistance of non-small cell lung cancer cells to cisplatin by targeting Bcl-2. Int. J. Mol. Med. 32, 593-598.
  24. Trebicka, J., Anadol, E., Elfimova, N., Strack, I., Roggendorf, M., Viazov, S., Wedemeyer, I., Drebber, U., Rockstroh, J., and Sauerbruch, T. (2013). Hepatic and serum levels of miR-122 after chronic HCVinduced fibrosis. J. Hepatol. 58, 234-239.
  25. Vermeulen, L., Wilde, G.D., Damme, P.V., Berghe, W.V., and Haegeman, G. (2003). Transcriptional activation of the $NF-{\kappa}B$ p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1). EMBO J. 22, 1313-1324.
  26. Weil, C., Nwankwo, C., Friedman, M., Kenet, G., Chodick, G., and Shalev, V. (2016). Epidemiology of hepatitis C virus infection in a large israeli health maintenance organization. J. Med. Virol. 56, 1907-1915.
  27. Xiao, Y., Tian, Q., He, J., Huang, M., Yang, C., and Gong, L. (2016). MiR-503 inhibits hepatocellular carcinoma cell growth via inhibition of insulin-like growth factor 1 receptor. Oncotargets Ther. 9, 3535-3544.
  28. Yin, Z.L., Wang, Y.L., Ge, S.F., Guo, T.T., Wang, L., Zheng, X.M., and Liu, J. (2015). Reduced expression of miR-503 is associated with poor prognosis in cervical cancer. Eur. Rev. Med. Pharmacol. Sci. 19, 4081- 4085.
  29. Yu, W., Coburn, C.A., Nair, A.G., Wong, M., Rosenblum, S.B., Zhou, G., Dwyer, M.P., Tong, L., Hu, B., Zhong, B., et al. (2016). Aryl or heteroaryl substituted aminal derivatives of HCV NS5A inhibitor MK- 8742. Bioorg. Med. Chem. Lett. 26, 3414-3420.
  30. Zeng, J., Chen, S., Li, N., Chen, L., Su, J., Niu, G., Zhu, S., and Liang, Y. (2015). Sasanquasaponin from Camellia oleifera Abel. induces apoptosis via Bcl-2, Bax and caspase-3 activation in HepG2 cells. Mol. Med. Rep. 12, 1997-2002.
  31. Zhao, Q., Yang, S.T., Wang, J.J., Zhou, J., Xing, S.S., Shen, C.C., Wang, X.X., Yue, Y.X., Song, J., Chen, M., et al. (2015). TNF alpha inhibits myogenic differentiation of C2C12 cells through $NF-{\kappa}B$ activation and impairment of IGF-1 signaling pathway. Biochem. Biophys. Res. Commun. 458, 790-795.
  32. Zhou, R., Gong, A.Y., Chen, D., Miller, R.E., Eischeid, A.N., and Chen, X.M. (2013). Histone deacetylases and $NF-{\kappa}B$ signaling coordinate expression of CX3CL1 in epithelial cells in response to microbial challenge by suppressing miR-424 and miR-503. PLos One 8, e65153.
  33. Park, K.J., Choi, S.H., Lee, S.Y., Hwang, S.B., and Lai, M.M. (2002). Nonstructural 5A protein of hepatitis C virus modulates tumor necrosis factor alpha-stimulated nuclear factor kappa B activation. J. Biol. Chem. 277, 13122-13128.

Cited by

  1. Viral Oncology: Molecular Biology and Pathogenesis vol.6, pp.12, 2017,
  2. Root Extract of Polygonum cuspidatum Siebold & Zucc. Ameliorates DSS-Induced Ulcerative Colitis by Affecting NF-kappaB Signaling Pathway in a Mouse Model via Synergistic Effects of Polydatin, Resveratrol, and Emodin vol.9, pp.1663-9812, 2018,