Tumor suppressor micro RNA miR-145 and onco micro RNAs miR-21 and miR-222 expressions are differentially modulated by Hepatitis B virus X protein in malignant hepatocytes

BMC Cancer

Manikankana Bandopadhyay⁴  Arup Banerjee⁴  Neelakshi Sarkar⁴  Rajesh Panigrahi²  Sibnarayan Datta³  Ananya Pal⁴  Shivram Prasad Singh¹  Avik Biswas⁴  Shekhar Chakrabarti⁵  Runu Chakravarty⁴ 

[1] Department of Gastroenterology, SCB medical college, Cuttack, India

[2] Present Address: Department of Pathology & Lab Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA

[3] Biodefence & Biodiversity Group, Defence Research Laboratory (DRDO), Tezpur, Assam, India

[4] ICMR Virus Unit, Kolkata, GB-4, 1st floor, ID & BG Hospital Campus, 57, Dr. S C Banerjee Road, Beliaghata, Kolkata West Bengal, 700010, India

[5] National Institute of Cholera and Enteric Diseases, Kolkata, India

关键词: microRNA;    HepG2.2.15;    HepG2;    Hepatocellular carcinoma;    HBx;   

Others  :  1121019
DOI  :  10.1186/1471-2407-14-721

 received in 2014-04-01, accepted in 2014-09-22,  发布年份 2014

【 摘 要 】

Background

Hepatitis B Virus (HBV) X protein (HBx) is known to be involved in the initiation and progression of hepatocellular carcinoma (HCC) through modulation of host gene response. Alterations in miRNA expressions are frequently noted in HCC. This study is aimed to examine the role of HBx protein in the modulation of oncogenic miRNA-21, miRNA-222 and tumor suppressor miRNA-145 in malignant hepatocytes.

Methods

Expressions of miRNA-21, miRNA-222 and miRNA-145 were measured in HepG2 cells transfected with HBx-plasmid (genotype D) and with full length HBV genome (genotype D) and also in stably HBV producing HepG2.2.15 cells using real time PCR. Their target mRNAs and proteins - PTEN, p27 and MAP3K - were analyzed by real time PCR and western blot respectively. miRNA expressions were measured after HBx/D mRNA specific siRNA treatment. The expressions of these miRNAs were analyzed in liver cirrhosis and HCC patients also.

Results

The study revealed a down-regulation of miRNA-21 and miRNA-222 expressions in HBx transfected HepG2 cells, pUC-HBV 1.3 plasmid transfected HepG2 cells as well as in HepG2.2.15 cells. Down regulation of miRNA-21 and miRNA-222 expression was observed in patient serum samples. Down regulation of miRNA-145 expression was observed in HepG2 cells transiently transfected with HBx and pUC-HBV1.3 plasmid as well as in patient samples but the expression of miRNA-145 was increased in HepG2.2.15 cells. Target mRNA and protein expressions were modulated in HepG2 cells and in HepG2.2.15 cell line consistent with the modulation of miRNA expressions.

Conclusion

Thus, HBx protein differentially modulated the expression of miRNAs. The study throws light into possible way by which HBx protein acts through microRNA and thereby regulates host functioning. It might suggest new therapeutic strategies against hepatic cancer.

【 授权许可】

   
2014 Bandopadhyay et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Lee YH, Yun Y: HBx protein of hepatitis B virus activates Jak1-STAT signaling. J Biol Chem 1998, 273:25510-25515.
• [2]Lee YI, Kang-Park S, Do SI, Lee YI: The hepatitis B virus-X protein activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade. J Biol Chem 2001, 276:16969-16977.
• [3]Haviv I, Shamay M, Doitsh G, Shaul Y: Hepatitis B virus pX targets TFIIB in transcription coactivation. Mol Cell Biol 1998, 18:1562-1569.
• [4]Feitelson MA, Lee J: Hepatitis B virus integration, fragile sites, and hepatocarcinogenesis. Cancer Lett 2007, 252(Suppl 2):157-170.
• [5]Wang XW, Forrester K, Yeh H, Feitelson MA, Gu JR, Harris C: Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3. Proc Natl Acad Sci U S A 1994, 91(Suppl 6):2230-2234.
• [6]Brechot C, Gozuacik D, Murakami Y, Paterlini-Brechot P: Molecular bases for the development of hepatitis B virus (HBV) related hepatocellular carcinoma (HCC). Semin Cancer Biol 2000, 10:211-231.
• [7]Kew MC: Increasing evidence that hepatitis B virus X gene protein and p53 protein may interact in the pathogenesis of hepatocellular carcinoma. Hepatology 1997, 25:1037-1038.
• [8]Murakami S: Hepatitis B virus X protein: structure, function and biology. Intervirology 1999, 42:81-99.
• [9]Yeh CT: Hepatitis B virus X protein: searching for a role in hepatocarcinogenesis. J Gastroenterol Hepatol 2000, 15:339-341.
• [10]Kew MC: Hepatitis B virus x protein in the pathogenesis of hepatitis B virus-induced hepatocellular carcinoma. J Gastroenterol Hepatol 2011, 26(Suppl 1):144-152.
• [11]Costa FF: Non-coding RNAs: new players in eukaryotic biology. Gene 2005, 357(Suppl 2):83-94.
• [12]Bartel DP: MicroRNAs: target recognition and regulatory functions. Cell 2009, 136(Suppl 2):215-233.
• [13]Calin GA, Croce CM: MicroRNA signatures in human cancers. Nat Rev Cancer 2006, 6(Suppl 11):857-866.
• [14]Wang B1, Majumder S, Nuovo G, Kutay H, Volinia S, Patel T, Schmittgen TD, Croce C, Ghoshal K, Jacob ST: Role of microRNA-155 at early stages of hepatocarcinogenesis induced by choline-deficient and amino acid-defined diet in C57BL/6 mice. Hepatology 2009, 50(Suppl 4):1152-1161.
• [15]Jiang J, Gusev Y, Aderca I, Mettler TA, Nagorney DM, Brackett DJ, Roberts LR, Schmittgen TD: Association of microRNA expression in hepatocellular carcinomas with hepatitis infection, cirrhosis, and patient survival. Clin Cancer Res 2008, 14(Suppl 2):14419-14427.
• [16]Huang YS, Dai Y, Yu XF, Bao SY, Yin YB, Tang M, Hu CX: Microarray analysis of microRNA expression in hepatocellular carcinoma and non-tumorous tissues without viral hepatitis. J Gastroenterol Hepatol 2008, 23:87-94.
• [17]Wang Y, Lee AT, Ma JZ, Wang J, Ren J, Yang Y, Tantoso E, Li KB, Ooi LL, Tan P, Lee CG: Profiling microRNA expression in hepatocellular carcinoma reveals microRNA-224 up-regulation and apoptosis inhibitor-5 as a microRNA-224-specific target. J Biol Chem 2008, 283:13205-13215.
• [18]Varnholt H, Drebber U, Schulze F, Wedemeyer I, Schirmacher P, Dienes HP, Odenthal M: MicroRNA gene expression profile of hepatitis C virus-associated hepatocellular carcinoma. Hepatology 2008, 47:1223-1232.
• [19]Galardi S, Mercatelli N, Giorda E, Massalini S, Frajese GV, Ciafrè SA, Farace M: miR-221 and miR-222 expression affects the proliferation potential of human prostate carcinoma cell lines by targeting p27 Kip1. J Biol Chem 2007, 282:23716-23724.
• [20]Wu G, Yu F, Xiao Z, Xu K, Xu J, Tang W, Wang J, Song E: Hepatitis B virus X protein down regulates expression of the miR-16 family in malignant hepatocytes in vitro. Br J Cancer 2011, 105:146-153.
• [21]Wang Y, Jiang L, Ji X, Yang B, Zhang Y, Fu XD: Hepatitis B viral RNA directly mediates down-regulation of the tumor suppressor microRNA mir-15a/mir-16-1 in hepatocytes. J Biol Chem 2013, 288(Suppl 25):18484-18493.
• [22]Kong G, Zhang J, Zhang S, Shan C, Ye L, Zhang X: Upregulated microRNA-29a by hepatitis B virus X protein enhances hepatoma cell migration by targeting PTEN in cell culture model. PLoS One 2011, 6(Suppl 5):e19518.
• [23]Wei X, Xiang T, Ren G, Tan C, Liu R, Xu X, Wu Z: miR-101 is down-regulated by the hepatitis B virus x protein and induces aberrant DNA methylation by targeting DNA methyl transferase 3A. Cell Signal 2013, 25(Supple 2):439-446.
• [24]Han Q, Zhang C, Zhang J, Tian Z: Involvement of activation of PKR in HBx-siRNA- mediated innate immune effects on HBV inhibition. PLoS One 2011, 6(Suppl 12):e27931.
• [25]Datta S, Banerjee A, Chandra PK, Biswas A, Panigrahi R, Mahapatra PK, Panda CK, Chakrabarti S, Bhattacharya SK, Chakravarty R: Analysis of hepatitis B virus X gene phylogeny, genetic variability and its impact on pathogenesis: Implications in Eastern Indian HBV carriers. Virology 2008, 382(Suppl 2):190-198.
• [26]Bouchard M, Giannakopoulos S, Wang EH, Tanese N, Schneider R: Hepatitis B virus HBx protein activation of cyclin A-cyclin dependent kinase 2 complexes and G1 transit via a Src kinase pathway. J Virol 2001, 75(Suppl 9):4247-4257.
• [27]Lee S, Tarn C, Wang WH, Chen S, Hullinger RL, Andrisani OM: Hepatitis B virus X protein differentially regulates cell cycle progression in X-transforming versus non transforming hepatocyte (AML12) cell lines. J Biol Chem 2002, 277:8730-8740.
• [28]Madden CR, Finegold M, Slagle B: Hepatitis B virus X protein acts as tumor promoter in development of diethylnitrosamine-induced pre neoplastic lesions. J Virol 2001, 75:3851-3858.
• [29]Madden R, Slagle BL: Stimulation of cellular proliferation by hepatitis B virus X protein. Dis Markers 2001, 17:153-157.
• [30]Lee YS, Dutta A: MicroRNAs in cancer. Annu Rev Pathol 2009, 4:199-227.
• [31]Xu X, Fan Z, Kang L, Han J, Jiang C, Zheng X, Zhu Z, Jiao H, Lin J, Jiang K, Ding L, Zhang H, Cheng L, Fu H, Song Y, Jiang Y, Liu J, Wang R, Du N, Ye Q: Hepatitis B virus X protein represses miRNA-148a to enhance tumorigenesis. J Clin Invest 2013, 123(Suppl 2):630-645.
• [32]Koff A: How to decrease p27Kip1 levels during tumor development. Cancer Cell 2006, 9:75-76.
• [33]Cl S, Nagel R, Egan DA, Schrier M, Mesman E, Mangiola A, Anile C, Maira G, Mercatelli N, Ciafrè SA, Farace MG, Agami R: Regulation of the p27Kip1 tumor suppressor by miR-221 and miR-222 promotes cancer cell proliferation. EMBO J 2007, 26:3699-3708.
• [34]Sirma H, Giannini C, Poussin K, Paterlini P, Kremsdorf D, Brechot C: Hepatitis B virus X mutants, present in hepatocellular carcinoma tissue abrogate both the anti proliferative and transactivation effects of HBx. Oncogene 1999, 18:4848-4859.
• [35]Qiao L, Leach K, McKinstry R, Gilfor D, Yacoub A, Park JS, Grant S, Hylemon PB, Fisher PB, Dent P: Hepatitis B virus X protein increases expression of p21(Cip-1/WAF1/MDA6) and p27(Kip-1) in primary mouse hepatocytes, leading to a reduced cell cycle progression. Hepatology 2001, 34:906-917.
• [36]Benn J, Schneider RJ: Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade. Proc Natl Acad Sci U S A 1994, 91:10350-10354.
• [37]Klein N, Schneider RJ: Activation of Src family kinases by hepatitis B virus HBx protein and coupled signaling to Ras. Mol Cell Biol 1997, 17:6427-6436.
• [38]Jiang X, Kanda T, Wu S, Nakamura M, Miyamura T, Nakamoto S, Banerjee A, Yokosuka O: Regulation of microRNA by hepatitis B virus infection and their possible association with control of innate immunity. World J Gastroenterol 2014, 20(Suppl 23):7197-7206.
• [39]Sells MA, Chen ML, Acs G: Production of hepatitis B virus particles in HepG2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci U S A 1987, 84(Suppl 4):1005-1009.
• [40]Chemin I, Zoulim F: Hepatitis B virus induced hepatocellular carcinoma. Cancer Lett 2009, 286:52-59.