Epigenetic repression of E-cadherin expression by hepatitis B virus x antigen in liver cancer - PubMed (original) (raw)

Epigenetic repression of E-cadherin expression by hepatitis B virus x antigen in liver cancer

A Arzumanyan et al. Oncogene. 2012.

Abstract

Loss of E-cadherin is associated with acquisition of metastatic capacity. Numerous studies suggest that histone deacetylation and/or hypermethylation of CpG islands in E-cadherin gene (CDH1) are major mechanisms responsible for E-cadherin silencing in different tumors and cancer cell lines. The hepatitis B virus (HBV)-encoded X antigen, HBx, contributes importantly to the development of hepatocellular carcinoma using multiple mechanisms. Experiments were designed to test if in addition to CDH1 hypermethylation HBx promotes epigenetic modulation of E-cadherin transcriptional activity through histone deacetylation and miR-373. The relationships between HBx, E-cadherin, mSin3A, Snail-1 and miR-373 were evaluated in HBx expressing (HepG2X) and control (HepG2CAT) cells by western blotting, immunoprecipitation (IP), chromatin IP as well as by immunohistochemical staining of liver and tumor tissue sections from HBV-infected patients. In HepG2X cells, decreased levels of E-cadherin and elevated levels of mSin3A and Snail-1 were detected. Reciprocal IP with anti-HBx and anti-mSin3A demonstrated mutual binding. Furthermore, HBx-mSin3A colocalization was detected by immunofluorescent staining. HBx downregulated E-cadherin expression by the recruitment of the mSin3A/histone deacetylase complex to the Snail-binding sites in human CDH1. Histone deacetylation inhibition by Trichostatin-A treatment restored E-cadherin expression. Mir-373, a positive regulator of E-cadherin expression, was downregulated by HBx in HepG2X cells and tissue sections from HBV-infected patients. Thus, histone deacetylation of CDH1 and downregulation of miR-373, together with the previously demonstrated hypermethylation of CDH1 by HBx, may be important for the understanding of HBV-related carcinogenesis.

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Conflict of interest statement

Conflict of interests

The authors declare that no conflict of interest exists and have no other disclosures to make.

Figures

Fig. 1

Fig. 1

Representative western blots with 50 μg of (A) total or (B) nuclear extracts of HepG2CAT (CAT) and HepG2X (X) cells. β-actin and lamin A are loading controls. Western blot analysis (C) and quantification (D) of protein extracts (50 μg) prepared from human hepatocytes after the treatment with transducible recombinant peptide 11R-HBx (+HBx) and control hepatocytes (-HBx). au = arbitrary units. This result was reproduced in each of three independent experiments with different batches of commercially available cells.

Fig. 2

Fig. 2

Representative staining for (A) HBx, Snail-1, mSin3A, and E-cadherin in consecutive sections from a block of a patient with HBV infected liver (x400). Representative staining for mSin3A (B) and Snail-1 (C) from uninfected human liver (top photo in B and C) and from a block of HCC (bottom photo in B and C) (x400).

Fig. 3

Fig. 3

Reciprocal immunoprecipitation of HepG2X and HepG2CAT protein extracts (A) with anti-mSin3A and western blot detection of HBx and (B) with anti-HBx and western blot detection of mSin3A. Lane 1 in panel A is a nuclear extract of HepG2X cells (70 μg). Immunofluorescent staining of HepG2X cells with anti-HBx (C), mSin3A (D), and DAPI (E). (F) is a merged image of HBx and mSin3A staining showing co-localization in the nucleus (arrow).

Fig. 4

Fig. 4

(A) Presented sequence for the human CDH1 promoter region was used for the design of primers and verified at AceView (NCBI). Primer binding sites for the target fragment (231 bp) are underlined, and the target fragment included the three E-boxes (1, 2, 3) that are Snail binding sites (Batlle et al., 2000). (B) ChIP assays of the E-cadherin promoter occupancy by mSin3A, HDAC1, Acetyl-H3 (Ac-H3), and RNA Polymerase II (RNA Pol II) in HepG2X and HepG2CAT cells. IP with IgG was used as a control. Primers for S15 (housekeeping gene) amplify a 361 bp fragment which was used as a control for ChIP with RNA Pol II. (C) Representative western blots showing levels of acetyl-H3 and E-cadherin after treatment of HepG2X and HepG2CAT cells with TSA.

Fig. 5

Fig. 5

(A) Expression of miR-373 in tumor and non-tumor liver tissues. Each bar represents the data collected from one patient. The difference in miRNA expression between tumor (T) and non-tumor (NT) was determined by qRT-PCR and determination of ΔΔCt, where ΔΔCt = ΔCt of miR-373 in tumor – ΔCt of miR-373 in non-tumor. U6 was used for normalization. Negative values indicate that miR-373 levels were higher in tumor compared to adjacent non-tumor. Positive values indicate that miR-373 levels were higher in NT (liver) compared to T. HBxAg NTa and HBxAg Ta: HBxAg staining in tumor (T) and nontumor (NT) is scored as follows: 0 = negative, 1 = up to 25% of cells stained positive, 2 = 25–50% of cells stained positive, 3 = > 50% cells stained positive. Dx NTb refers to diagnosis of lesions in nontumor liver. These are as follows: 0 = no significant lesions, 1 = chronic hepatitis, 2 = cirrhosis. edmon Tc refers to the Edmondson classification of cellular differentiation within tumor nodules. They are as follows: 1 = Edmondson I–II, 2 = Edmondson II, 3 = Edmondson II–III, 4 = Edmondson III. Vein inv Td refers to venous invasion of the tumor nodule, where 0 = no evidence for invasion, and 1 = presence of venous invasion. Encap Te refers to tumor encapsulation, where 0 = none and 1 = encapsulation. (B) Western blot detection of E-cadherin with 50 μg of protein extracts from HepG2X cells (−) and from HepG2X cells transiently transfected with miR-373 precursor (+).

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