Background: The molecular interactions of hepatitis C virus (HCV) with hepatic tissue have yet to be completely elucidated and understood. The purpose of this study was to compare differential gene expression patterns in the livers of non-transgenic and transgenic mouse model expressing HCV structural proteins Core, Envelope 1 (E1) and Envelope 2 (E2) using complementary DNA (cDNA) microarrays. Results: Total RNA extracted from the livers of HCV transgenic and non-transgenic mice was analyzed with cDNA microarray and differentially expressed genes confirmed by real-time RT-PCR. Relative expression ratios of individual genes were determined by comparing hybridization of Cy5-labelled cDNA from transgenic mouse livers and Cy3-labelled cDNA from non-transgenic mouse livers. The spot array images were quantified using QuantArray software and the outlier spots was normalized and filtered using five different criteria. 15,297 genes were analyzed using three different analytical methods. Depending on these methods, twenty-one genes were found to be differentially expressed at a statistically significant level. From these, 6 genes had a consistent differential expression. Several genes were directly involved in lipid metabolism and lipid β -oxidation. 5-azacytidine induced gene 2 (AZ2), which is involved in the methylation of genes was down regulated in HCV transgenic mice. Altered transcript levels of these 6 genes were confirmed by real-time RT-PCR analysis. Conclusion: Interactions between HCV and hepatocytes not only involve lipid metabolism and redox balance, but this interaction may also influence DNA methylation, indicating a potential association with the development of hepatocellular carcinoma.
KeywordsMicroarrayHCVHepatocellular Carcinoma
Cohen, J. (1999) The scientific challenge of hepatitis C. Science, 285, 26-30. doi:10.1126/science.285.5424.26
Rosen, H.R. and Gretch, D.R. (1999) Hepatitis C virus: Current understanding and prospects for future therapies. Molecular Medicine Today, 5, 393-399. doi:10.1016/S1357-4310(99)01523-3
Colombo, M. and Covini, G. (1995) Hepatitis C virus and hepatocellular carcinoma. Clinical and Experimental Rheumatology, 13, S23-S27.
Liang, T.J. and Heller, T. (2004) Pathogenesis of hepatitis C-associated hepatocellular carcinoma. Gastroenterology, 127, S62-S71. doi:10.1053/j.gastro.2004.09.017
Alter, M.J. (1997) Epidemiology of hepatitis C. Hepatology, 26, 62S-65S. doi:10.1002/hep.510260711
Gasiorowicz, M., Hurie, M., Russell, A., Hoxie, N. and Vergeront, J. (2006) Epidemiologic trends in infection, mortality, and transplants related to hepatitis C in Wisconsin. WMJ, 105, 34-39.
Ray, R.B., Lagging, L.M., Meyer, K. and Ray, R. (1996) Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype. Journal of Virology, 70, 4438-4443.
Honda, A., Arai, Y., Hirota, N., Sato, T., Ikegaki, J., Koizumi, T., et al. (1999) Hepatitis C virus structural proteins induce liver cell injury in transgenic mice. Journal of Medical Virology, 59, 281-289. doi:10.1002/(SICI)1096-9071(199911)59:3 3.0.CO;2-S
Lerat, H., Honda, M., Beard, M.R., Loesch, K., Sun, J., Yang, Y., et al. (2002) Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus. Gastroenterology, 122, 352-365. doi:10.1053/gast.2002.31001
Moriya, K., Fujie, H., Yotsuyanagi, H., Shintani, Y., Tsutsumi, T., Matsuura, Y., et al. (1997) Subcellular localization of hepatitis C virus structural proteins in the liver of transgenic mice. Japanese Journal of Medical Science & Biology, 50, 169-177.
Moriya, K., Fujie H, Shintani, Y., Yotsuyanagi, H., Tsutsumi, T., Ishibashi, K., et al. (1998) The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nature Medicine, 4, 1065-1067. doi:10.1038/2053
Moriya, K., Nakagawa, K., Santa, T., Shintani, Y., Fujie, H., Miyoshi, H., et al. (2001) Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis. Cancer Research, 61, 4365-4370.
Naas, T., Ghorbani, M., Alvarez-Maya, I., Lapner, M., Kothary, R., De, R.Y., et al. (2005) Characterization of liver histopathology in a transgenic mouse model expressing genotype 1a hepatitis C virus core and envelope proteins 1 and 2. Journal of General Virology, 86, 2185-2196. doi:10.1099/vir.0.80969-0
Sola, R., Alvarez, M.A., Balleste, B., Montoliu, S., Rivera, M., Miquel, M., et al. (2006) Probability of liver cancer and survival in HCV-related or alcoholic-decompensated cirrhosis. A study of 377 patients. Liver International, 26, 62-72. doi:10.1111/j.1478-3231.2005.01181.x
Alonzi, T., Agrati, C., Costabile, B., Cicchini, C., Amicone, L., Cavallari, C., et al. (2004) Steatosis and intrahepatic lymphocyte recruitment in hepatitis C virus transgenic mice. Journal of General Virology, 85, 1509-1520. doi:10.1099/vir.0.19724-0
Feitelson, M.A. and Larkin, J.D. (2001) New animal models of hepatitis B and C. ILAR Journal, 42, 127-138.
Ren, J.Y., Cheng, G.X., Kong, X.F., Chen, J.Q., Zhou, R.J. and Lu, Z.M. (2005) Establish a transgenic mice model harboring structural genes of hepatitis C virus. Chinese Journal of Hepatology, 13, 501-504.
Wakita, T. (1998) Construction of HCV transgenic mice with Cre/loxP switching expression system. Journal of Virology, 48, 9-18.
Elshourbagy, N.A., Walker, D.W., Paik, Y.K., Boguski, M.S., Freeman, M., Gordon, J.I., et al. (1987) Structure and expression of the human apolipoprotein A-IV gene. The Journal of Biological Chemistry, 262, 7973-7981.
Windmueller, H.G. and Spaeth, A.E. (1985) Regulated biosynthesis and divergent metabolism of three forms of hepatic apolipoprotein B in the rat. The Journal of Lipid Research, 26, 70-81.
Kono, Y., Hayashida, K., Tanaka, H., Ishibashi, H. and Harada, M. (2003) High-density lipoprotein binding rate differs greatly between genotypes 1b and 2a/2b of hepatitis C virus. Journal of Medical Virology, 70, 42-48. doi:10.1002/jmv.10372
Wu, A.L. and Windmueller, H.G. (1981) Variant forms of plasma apolipoprotein B. Hepatic and intestinal biosynthesis and heterogeneous metabolism in the rat. The Journal of Biological Chemistry, 256, 3615-3618.
Wu, A.L. and Windmueller, H.G. (1978) Identification of circulating apolipoproteins synthesized by rat small intestine in vivo. The Journal of Biological Chemistry, 253, 2525-2528.
Barba, G., Harper, F., Harada, T., Kohara, M., Goulinet, S., Matsuura, Y., et al. (1997) Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets. Proceedings of the National Academy of Sciences USA, 94, 1200-1205. doi:10.1073/pnas.94.4.1200
Perlemuter, G., Sabile, A., Letteron, P., Vona, G., Topilco, A., Chretien, Y., et al. (2002) Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: A model of viral-related steatosis. FASEB Journal, 16, 185-194. doi:10.1096/fj.01-0396com
Sabile, A., Perlemuter, G., Bono, F., Kohara, K., Demaugre, F., Kohara, M., et al. (1999) Hepatitis C virus core protein binds to apolipoprotein AII and its secretion is modulated by fibrates. Hepatology, 30, 1064-1076. doi:10.1002/hep.510300429
Korenaga, M., Wang, T., Li, Y., Showalter, L.A., Chan, T., Sun, J., et al. (2005) Hepatitis C virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ROS) production. The Journal of Biological Chemistry, 280, 37481-37488. doi:10.1074/jbc.M506412200
Tso, P., Chen, Q., Fujimoto, K., Fukagawa, K. and Sakata, T. (1995) Apolipoprotein A-IV: A circulating satiety signal produced by the small intestine. Obesity Research, 3, 689S-695S. doi:10.1002/j.1550-8528.1995.tb00487.x
Soardo, G., Pirisi, M., Fonda, M., Fabris, C., Falleti, E., Toniutto, P., et al. (1995) Changes in blood lipid composition and response to interferon treatment in chronic hepatitis C. Journal of Interferon & Cytokine Research, 15, 705-712. doi:10.1089/jir.1995.15.705
Cheng, Y., Dharancy, S., Malapel, M. and Desreumaux, P. (2005) Hepatitis C virus infection down-regulates the expression of peroxisome proliferator-activated receptor alpha and carnitine palmitoyl acyl-CoA transferase 1A. World Journal of Gastroenterology, 11, 7591-7596.
Kuramitsu, Y. and Nakamura, K. (2005) Current progress in proteomic study of hepatitis C virus-related human hepatocellular carcinoma. Expert Review of Proteomics, 2, 589-601. doi:10.1586/14789450.2.4.589
Chekhun, V.F., Kulik, G.I., Yurchenko, O.V., Tryndyak, V.P., Todor, I.N., Luniv, L.S., et al. (2006) Role of DNA hypomethylation in the development of the resistance to doxorubicin in human MCF-7 breast adenocarcinoma cells. Cancer Letters, 231, 87-93. doi:10.1016/j.canlet.2005.01.038
Tryndyak, V.P., Kovalchuk, O. and Pogribny, I.P. (2006) Loss of DNA methylation and histone H4 lysine 20 trimethylation in human breast cancer cells is associated with aberrant expression of DNA methyltransferase 1, Suv4-20h2 histone methyltransferase and methyl-binding proteins. Cancer Biology & Therapy, 5, 65-70. doi:10.4161/cbt.5.1.2288
Pogribny, I.P., Ross, S.A., Wise, C., Pogribna, M., Jones, E.A., Tryndyak, V.P., et al. (2006) Irreversible global DNA hypomethylation as a key step in hepatocarcinogenesis induced by dietary methyl deficiency. Mutation Research, 593, 80-87. doi:10.1016/j.mrfmmm.2005.06.028
Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25, 402-408.