Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

HBx-mediated miR-21 upregulation represses tumor-suppressor function of PDCD4 in hepatocellular carcinoma

Oncogene volume 32, pages 3296–3305 (2013)Cite this article

Subjects

Abstract

The hepatitis B virus (HBV) X protein (HBx) has a key role in the molecular pathogenesis of HBV-related hepatocellular carcinoma (HCC). However, the mechanism of HBx-mediated hepatocarcinogenesis remains to be elucidated. In this study, we aimed to better understand the effects of HBx on gene-expression profiles that participate in hepatocarcinogenesis and the mechanism by which HBx regulates these genes. Differentially expressed genes between L02-HBx and L02-Vector control cells were identified by microarray and validated using quantitative real-time PCR. HBx upregulates 456 genes and downregulates 843 genes, including programmed cell death 4 (PDCD4). PDCD4 was downregulated in clinical HCC specimens and the downregulation of PDCD4 in HCC is correlated with HBx. Furthermore, overexpression experiments in HCC cells proved that PDCD4 has strong tumor-suppressive effects both in vitro and in vivo, and may induce cell apoptosis to suppress the development of HCC. HBx induces expression of DNA methyltransferases (DNMTs), but failed to change the methylation status of the PDCD4 promoter. HBx downregulates PDCD4 expression at least partially through miR-21. Taken together, this study reported for the first time that HBx downregulates PDCD4 and upregulates miR-21 expression. The overexpression of PDCD4 could suppress tumorigenicity. The deregulation of PDCD4 by HBx through miR-21 represents a potential novel mechanism of the downregulation of PDCD4 in HBV-related HCC and provides new insights into HCC development.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

Abbreviations

DNMTs:

DNA methyltransferases

HBV:

hepatitis B virus

HBx:

hepatitis B virus X protein

HCC:

hepatocellular carcinoma

miRNA:

microRNA

MSP:

methylation-specific PCR

PDCD4:

programmed cell death 4

qPCR:

quantitative real-time PCR

siRNA:

small interfering RNA

TSG:

tumor-suppressor gene

References

  1. Tan A, Yeh SH, Liu CJ, Cheung C, Chen PJ . Viral hepatocarcinogenesis: from infection to cancer. Liver Int 2008; 28: 175–188.

    Article  CAS  Google Scholar 

  2. Lupberger J, Hildt E . Hepatitis B virus-induced oncogenesis. World J Gastroenterol 2007; 13: 74–81.

    Article  CAS  Google Scholar 

  3. Wang Y, Cui F, Lv Y, Li C, Xu X, Deng C et al. HBsAg and HBx knocked into the p21 locus causes hepatocellular carcinoma in mice. Hepatology 2004; 39: 318–324.

    Article  CAS  Google Scholar 

  4. Hanahan D, Weinberg R . The hallmarks of cancer. Cell 2000; 100: 57–70.

    Article  CAS  Google Scholar 

  5. Buendia A . Genetics of hepatocellular carcinoma. Semin Cancer Biol 2000; 10: 185–200.

    Article  CAS  Google Scholar 

  6. Ozaki I, Mizuta T, Zhao G, Yotsumoto H, Hara T, Kajihara S et al. Involvement of the Ets-1 gene in overexpression of matrilysin in human hepatocellular carcinoma. Cancer Res 2000; 60: 6519–6525.

    CAS  PubMed  Google Scholar 

  7. Huang JZ, Xia SS, Ye QF, Jiang HY, Chen ZH . Effects of p16 gene on biological behavious in hepatocellular carcimoma cells. World J Gastroenterol 2003; 9: 84–88.

    Article  CAS  Google Scholar 

  8. Zhang H, Ozaki I, Mizuta T, Hamajima H, Yasutake T, Eguchi Y et al. Involvement of programmed cell death 4 in transforming growth factor-β1-induced apoptosis in human hepatocellular carcinoma. Oncogene 2006; 25: 6101–6112.

    Article  CAS  Google Scholar 

  9. Soejima K, Fang W, Rollins BJ . DNA methyltransferase 3b contributes to oncogenic transformation induced by SV40T antigen and activated Ras. Oncogene 2003; 22: 4723–4733.

    Article  CAS  Google Scholar 

  10. Tsai CN, Tsai CL, Tse KP, Chang HY, Chang YS . The Epstein-Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases. Proc Natl Acad Sci USA 2002; 99: 10084–10089.

    Article  CAS  Google Scholar 

  11. Lok AS, Heathcote EJ, Hoofnagle JH . Management of hepatitis B: 2000-summary of a workshop. Gastroenterology 2001; 120: 1828–1853.

    Article  CAS  Google Scholar 

  12. Lee JO, Kwun HJ, Jung JK, Choi KH, Min DS, Jang KL . Hepatitis B virus X protein represses E-cadherin expression via activation of DNA methyltransferase 1. Oncogene 2005; 24: 6617–6625.

    Article  CAS  Google Scholar 

  13. Park IY, Sohn BH, Yu E, Suh DJ, Chung YH, Lee JH et al. Aberrant epigenetic modifications in hepatocarcinogenesis induced by hepatitis B virus X protein. Gastroenterology 2007; 132: 1476–1494.

    Article  CAS  Google Scholar 

  14. Wang Y, Lu Y, Toh ST, Sung WK, Tan P, Chow P et al. Lethal-7 is down-regulated by the hepatitis B virus x protein and targets signal transducer and activator of transcription 3. J Hepatol 2010; 53: 57–66.

    Article  CAS  Google Scholar 

  15. Murakami Y, Yasuda T, Saigo K, Urashima T, Toyoda H, Okanoue T et al. Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues. Oncogene 2005; 25: 2537–2545.

    Article  Google Scholar 

  16. Ladeiro Y, Couchy G, Balabaud C, Bioulac-Sage P, Pelletier L, Rebouissou S et al. MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations. Hepatology 2008; 47: 1955–1963.

    Article  CAS  Google Scholar 

  17. Hajjou M, Norel R, Carver R, Marion P, Cullen J, Rogler LE et al. cDNA microarray analysis of HBV transgenic mouse liver identifies genes in lipid biosynthetic and growth control pathways affected by HBV. J Med Virol 2005; 77: 57–65.

    Article  CAS  Google Scholar 

  18. Okabe H, Satoh S, Kato T, Kitahara O, Yanagawa R, Yamaoka Y et al. Genome-wide analysis of gene expression in human hepatocellular carcinomas using cDNA microarray. Cancer Res 2001; 61: 2129–2137.

    CAS  Google Scholar 

  19. Gao F, Wang X, Zhu F, Wang Q, Zhang X, Guo C et al. PDCD4 gene silencing in gliomas is associated with 5'CpG island methylation and unfavourable prognosis. J Cell Mol Med 2009; 13: 4257–4267.

    Article  CAS  Google Scholar 

  20. Fan H, Zhao Z, Quan Y, Xu J, Zhang J, Xie W . DNA methyltransferase 1 knockdown induces silenced CDH1 gene reexpression by demethylation of methylated CpG in hepatocellular carcinoma cell line SMMC-7721. Eur J Gastroenterol Hepatol 2007; 19: 952–961.

    Article  CAS  Google Scholar 

  21. Asangani I, Rasheed S, Nikolova D, Leupold J, Colburn N, Post S et al. MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene 2007; 27: 2128–2136.

    Article  Google Scholar 

  22. Frankel LB, Christoffersen NR, Jacobsen A, Lindow M, Krogh A, Lund AH . Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells. J Biol Chem 2008; 283: 1026–1033.

    Article  CAS  Google Scholar 

  23. Zhang X, Liu S, Hu T, He Y, Sun S . Up-regulated microRNA-143 transcribed by nuclear factor kappa B enhances hepatocarcinoma metastasis by repressing fibronectin expression. Hepatology 2009; 50: 490–499.

    Article  CAS  Google Scholar 

  24. 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: e19518.

    Article  CAS  Google Scholar 

  25. Matsuhashi S, Yoshinaga H, Yatsuki H, Tsugita A, Hori K . Isolation of a novel gene from a human cell line with Pr-28 MAb which recognizes a nuclear antigen involved in the cell cycle. Res Commun Biochem Cell Mol Biol 1997; 1: 109–120.

    CAS  Google Scholar 

  26. Yoshinaga H, Matsuhashi S, Fujiyama C, Masaki Z . Novel human PDCD4 (H731) gene expressed in proliferative cells is expressed in the small duct epithelial cells of the breast as revealed by an anti-H731 antibody. Pathol Int 1999; 49: 1067–1077.

    Article  CAS  Google Scholar 

  27. Wang Q, Sun Z, Yang H . Downregulation of tumor suppressor Pdcd4 promotes invasion and activates both β-catenin/Tcf and AP-1-dependent transcription in colon carcinoma cells. Oncogene 2007; 27: 1527–1535.

    Article  Google Scholar 

  28. Zhang S, Li J, Jiang Y, Xu Y, Qin C . Programmed cell death 4 (PDCD4) suppresses metastastic potential of human hepatocellular carcinoma cells. J Exp Clin Cancer Res 2009; 28: 1–11.

    Article  Google Scholar 

  29. Hilliard A, Hilliard B, Zheng SJ, Sun H, Miwa T, Song W et al. Translational regulation of autoimmune inflammation and lymphoma genesis by programmed cell death 4. J Immunol 2006; 177: 8095–8102.

    Article  CAS  Google Scholar 

  30. Schmid T, Jansen AP, Baker AR, Hegamyer G, Hagan JP, Colburn NH . Translation inhibitor Pdcd4 is targeted for degradation during tumor promotion. Cancer Res 2008; 68: 1254–1260.

    Article  CAS  Google Scholar 

  31. Jansen AP, Camalier CE, Colburn NH . Epidermal expression of the translation inhibitor programmed cell death 4 suppresses tumorigenesis. Cancer Res 2005; 65: 6034–6041.

    Article  CAS  Google Scholar 

  32. Zhang X, Wang X, Song X, Liu C, Shi Y, Wang Y et al. Programmed cell death 4 enhances chemosensitivity of ovarian cancer cells by activating death receptor pathway in vitro and in vivo. Cancer Sci 2010; 10: 2163–2170.

    Article  Google Scholar 

  33. Lankat-Buttgereit B, Goke R . Programmed cell death protein 4 (pdcd4): A novel target for antineoplastic therapy? Biol Cell 2003; 95: 515–520.

    Article  CAS  Google Scholar 

  34. Ozturk M . Genetic aspects of hepatocellular carcinogenesis. Semin Liver Dis 1999; 19: 235–242.

    Article  CAS  Google Scholar 

  35. Kusano N, Shiraishi K, Kubo K, Oga A, Okita K, Sasaki K . Genetic aberrations detected by comparative genomic hybridization in hepatocellular carcinomas: their relationship to clinicopathological features. Hepatology 1999; 29: 1858–1862.

    Article  CAS  Google Scholar 

  36. Yu MW, Chen CJ . Hepatitis B and C viruses in the development of hepatocellular carcinoma. Crit Rev Oncol Hematol 1994; 17: 71–91.

    Article  CAS  Google Scholar 

  37. Zheng DL, Zhang L, Cheng N, Xu X, Deng Q, Teng XM et al. Epigenetic modification induced by hepatitis B virus X protein via interaction with de novo DNA methyltransferase DNMT3A. J Hepatol 2009; 50: 377–387.

    Article  CAS  Google Scholar 

  38. Li J, Fu H, Xu C, Tie Y, Xing R, Zhu J et al. miR-183 inhibits TGF-β1-induced apoptosis by downregulation of PDCD4 expression in human hepatocellular carcinoma cells. BMC Cancer 2010; 10: 354–363.

    Article  Google Scholar 

  39. Dorrello NV, Peschiaroli A, Guardavaccaro D, Colburn NH, Sherman NE, Pagano M . S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth. Science 2006; 314: 467–471.

    Article  CAS  Google Scholar 

  40. Luber B, Lauer U, Weiss L, Höhne M, Hofschneider PH, Kekulé AS . The hepatitis B virus transactivator HBx causes elevation of diacylglycerol and activation of protein kinase C. Res Virol 1993; 144: 311–321.

    Article  CAS  Google Scholar 

  41. Tanaka Y, Kanai F, Ichimura T, Tateishi K, Asaoka Y, Guleng B et al. The hepatitis B virus X protein enhances AP-1 activation through interaction with Jab1. Oncogene 2006; 25: 633–642.

    Article  CAS  Google Scholar 

  42. Talotta F, Cimmino A, Matarazzo MR, Casalino L, De Vita G, D'Esposito M et al. An autoregulatory loop mediated by miR-21 and PDCD4 controls the AP-1 activity in RAS transformation. Oncogene 2009; 28: 73–84.

    Article  CAS  Google Scholar 

  43. Yang HS, AP Jansen, Nair R, Shibahara K, Verma AK, Cmarik JL et al. A novel transformation suppressor, Pdcd4, inhibits AP-1 transactivation but not NF-kappaB or ODC transactivation. Oncogene 2001; 20: 669–676.

    Article  CAS  Google Scholar 

  44. Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 2005; 33: e179.

    Article  Google Scholar 

  45. Si M, Zhu S, Wu H, Lu Z, Wu F, Mo Y . miR-21-mediated tumor growth. Oncogene 2006; 26: 2799–2803.

    Article  Google Scholar 

  46. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB . Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996; 93: 9821–9826.

    Article  CAS  Google Scholar 

  47. Leupold JH, Asangani IA, Mudduluru G, Allgayer H . Promoter cloning and characterization of the human programmed cell death protein 4 (pdcd4) gene: evidence for ZBP-89 and Sp-binding motifs as essential Pdcd4 regulators. Biosci Rep 2012; 32: 281–297.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by The National Natural Science Foundation of China, no. 30470950, National 973 Basic Research Program of China 2013CB911302, National Natural Science Foundation of China, no. 91229107 and in part by Innovative Scientific Research Projects for College Graduates of Jiangsu Province, no. CXZZ_0137. We thank Professor Guan Xinyuan in the Univrsity of Hong Kong for providing the HBx construct, Dr Olubunmi Afonja in New York University for providing the PDCD4 construct and Dr Heike Allgayer in the University of Heidelberg for providing the PDCD4 promoter constructs.

Author information

Authors and Affiliations

  1. Department of Medical Genetics and Developmental Biology, Medical School of Southeast University, The Key Laboratory of Developmental Genes and Human Diseases, Ministry of Education, Southeast University, Nanjing, China

    X Qiu, F Qiao, Y Song, Y Lao, J Hu & H Fan

  2. Department of Clinical Oncology, The University of Hong Kong, Hong Kong, China

    S Dong

  3. The First Affiliated Hospital with Nanjing Medical University, Nanjing, China

    S Lu

  4. State Key Laboratory of Oncology in Southern China, Cancer Center, Sun Yat-sen University, Guangzhou, China

    Y Li & T Zeng

  5. Department of Immunology, Shandong University School of Medicine, Jinan, China

    L Zhang

  6. The Affiliated Zhongda Hospital of Southeast University, Nanjing, China

    L Zhang

Authors
  1. X Qiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. F Qiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. S Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Y Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Y Lao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Y Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. T Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. J Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. L Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. L Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. H Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to H Fan.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Qiu, X., Dong, S., Qiao, F. et al. HBx-mediated miR-21 upregulation represses tumor-suppressor function of PDCD4 in hepatocellular carcinoma. Oncogene 32, 3296–3305 (2013). https://doi.org/10.1038/onc.2013.150

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2013.150

Keywords

This article is cited by

Search

Advanced search

Quick links