Mutations in the C-terminus of the X protein of hepatitis B virus regulate Wnt-5a expression in hepatoma Huh7 cells: cDNA microarray and proteomic analyses

Abstract

Background: The hepatitis B virus x gene (HBx) is a promiscuous transactivator implicated in the development of hepatocellular carcinoma (HCC). The present study was designed to investigate the molecular events regulated by HBx.

Methods: Genomic and proteomic expression profiling was performed in Huh7 HCC cells transfected with HBx mutants with a C-terminal deletion. The gene and protein expression of wingless-type murine-mammary-tumour virus (MMTV) integration site family, member 5A (Wnt-5a) was validated by analyses of reverse transcription-polymerase chain reaction (RT-PCR), real-time RT-PCR, western blot and immunohistochemistry.

Results: Differentially expressed genes and proteins were found in the transfected Huh7 HCC cells; most of them were involved in transcriptional regulation, although others including oncogenes or tumor suppressor genes, and molecules involved in cell junctions, signal transduction pathways, metabolism or the immune response were also observed. The expression of the Wnt-5a gene was elevated >10-fold in Huh7 cells transfected with the HBx3'-30 amino acid deletion mutant. However, the expression was downregulated by the transfection with the HBx3'-40 amino acid deletion mutant. The changes in Wnt-5a expression were also observed in human HCC tissues, compared with corresponding non-cancerous liver tissues. A negative correlation was found between the expression of Wnt-5a and HBx COOH mutations in HCC tissues.

Conclusions: HBx mutants may participate in the development and progression of HCC, at least in part through the Wnt-5a pathway.

Fig. 1.

Detection of HBx deletion mutant plasmid. (A) The fragment size was matched the predicted fragment by restriction digest, suggesting that the correct plasmid construct was present. (B) The sequencing results have been confirmed theirs success constructed. (C) The Neo gene amplified by PCR. (D) The full-length HBx and the four truncated HBx expression vectors was expressed the hepatitis B X protein antigen by western blot, the purified bands had a molecular size of ∼17 kDa, corresponding with the size of the HBx protein.

Fig. 2.

Bar graphs showing the profile of the differentially expressed genes in cells transfected with the HBx3′-30 and HBx3′-40 mutants. The differentially expressed genes are involved in transcriptional regulation, cytoskeletal integrity and adhesion, signal transduction, metabolism, apoptosis and the immune response.

Fig. 3.

Two-dimensional gel electrophoresis of protein expression in Huh7 cells transfected with the HBx3′-30 or HBx3′-40 mutant, or with full-length HBx. The red arrow indicates overexpressed protein, and green arrow indicates decreased protein expression.

Fig. 4.

Wnt-5a mRNA and protein expression in Huh7 cells transfected with the HBx3′-30 or HBx3′-40 mutant, and in human clinical HCC and corresponding non-cancerous liver tissue samples. (A) RT–PCR. Wnt-5a mRNA was upregulated in HBx3′-30-transfected Huh7 cells but downregulated in HBx3′-40-transfected Huh7 cells, compared with cells expressing full-length HBx. Wnt-5a mRNA expression varies in human HCCs when compared with their corresponding non-cancerous liver tissues. (B) Real-time RT–PCR. No statistically significant difference in Wnt-5a mRNA expression was found between HCC and the corresponding non-cancerous liver tissues. (C) Western blotting. The expression of Wnt-5a protein was upregulated in HBx3′-30-transfected Huh7 cells, but was not significantly different in HBx3′-40-transfected Huh7 cells, compared with cells transfected with full-length HBx. (D) The protein was present at significantly lower levels in the tumor tissues than in the corresponding non-cancerous liver tissue (P < 0.01); N, non-tumor; T, HCC; Bars, mean; columns, standard deviation.