[Hepatitis B virus X protein induces malignant transformation tendency of Chang liver cells involving p16(INK4a) hypermethylation].

OBJECTIVE: To study the relationship between hepatitis B virus X (HBx) protein and DNA methylation of p16(INK4a) and the role of HBx in the carcinogenesis of hepatocellular carcinoma. METHODS: Eukaryonic expression vectors pcDNA3.1B-HBx and pcDNA3.1B were transduced into Chang liver cells by using Lipofectamine 2000 to establish the Chang-HBx liver cell line (HBx expression) and Chang-vector liver cell line (non-HBx expression). RT-PCR and Western blot were used to test the expression of p16(INK4a) in the two cell lines. The level of p16(INK4a) promoter methylation was tested by methylation specific PCR (MSP). The proliferation curves were drew by CCK-8, and S-phase in cell cycle and apoptosis were observed by flow cytometry. RESULTS: Hypermethylation of p16 can be mediated by HBx, which decreases the expression of mRNA and protein of p16. Chang-HBx cells grow faster. Chang-HBx cells have much higher S-phase population (28.96% vs. 21.53%, P<0.001; 28.96% vs. 21.5%, P<0.001) and lower apoptosis rate (2.71% vs. 3.69%, P<0.001; 2.71% vs. 3.36%, P<0.001) than Chang-vector cells and Chang cells respectively. CONCLUSION: p16(INK4a) expression was repressed by HBx protein via DNA methylation of p16(INK4a), which can induce the malignant transformation tendency of Chang cells.

Up-regulation of RNF187 induces hepatocellular carcinoma cell epithelial to mesenchymal transitions.

Ring finger protein 187 (RNF187) has been identified to be a co-activator linking c Jun to Ras signaling. However, the expression and function of RNF187 in hepatocellular carcinomas (HCC) remains unclear. Here, we tried to determine the expression and roles of RNF187 in hepatocellular carcinomas (HCC).The expression of RNF187 was determined in HCC tissues and cell lines, and we found that RNF187 expressed highly in HCC tissues compared with the corresponding adjacent liver tissues both in mRNA and protein level, which was consistent with the result of immunohistochemistry on HCC tissue microarrays. In HCC cell lines, the level of RNF187 was positively associated with the HCC cells metastatic potential. By the RNF187 interference and cDNA transfection, we showed that the high level of RNF187 induced the HCC cells invasion and metastasis both in vitro and in vivo, as well as the high ability of colony formation.Mechanistically, we detected the high level of RNF187 promoted cell scatter by inducing epithelial-mesenchymal transition (EMT). Clinically, the high level of RNA187 was significantly correlated with a malignant phenotype, including larger tumor size, multiple tumors, and microvascular invasion. Importantly, high level of RNF187 correlated with HCC patients' shorter OS and lower disease free survival rates than those with low level of RNF187. Our results revealed that elevated expression of RNF187 induced hepatocellular carcinoma cell epithelial to mesenchymal transitions, and represented a novel marker for predicting the poor prognosis of HCC.

Genomic aberrations in the HTPAP promoter affect tumor metastasis and clinical prognosis of hepatocellular carcinoma.

We previously reported that the intronic tagSNP +357G/C in the metastasis suppressor HTPAP is associated with metastasis and prognosis of hepatocellular carcinoma (HCC). The aim of this study was to investigate whether SNPs in the HTPAP promoter modulate HTPAP expression and prognosis of HCC. Genomic DNA from 572 microdissected HCCs were genotyped by pyrosequencing and verified by direct sequencing. Haplotype blocks were analyzed. Reporter plasmids were constructed and transfected into HCC cell lines. Transcriptional activities of plasmids were analyzed by dual-luciferase reporter systems. HTPAP expression was measured by real-time quantitative PCR, western blots, and tissue microarrays. Invasion was assessed by Matrigel assays. The prognostic values of HTPAP promoter SNPs in HCC were evaluated by Kaplan-Meier and Cox regression analyses. We identified six SNPs, including -1053A/G and +64G/C, in the HTPAP promoter. The SNPs were in complete linkage disequilibrium, resulting in three promoter haplotypes (promoter I:-1053AA/+64GG, promoter II: -1053AG/+64GC, and promoter III: -1053GG/+64CC). Promoter I manifested the highest luciferase index (p<0.005). However, no significant difference was observed between promoters II and III. We consistently found that HTPAP mRNA and protein levels were significantly higher in promoter I than that of promoter II+III (p<0.001). Invasion was increased in HCC cells transfected with promoters II+III compared to those transfected with promoter I (p<0.05). The HTPAP promoter II+III haplotype was associated with significantly increased metastasis compared to that of promoter I (p = 0.023). The postoperative five-year overall survival of patients with promoters II+III was lower than that of patients with promoter I (p = 0.006). Multivariate analysis showed that the promoter II+III haplotype was an adverse prognostic marker in HCC. The genetic variants at loci -1053 and +64 of the HTPAP promoter affect the expression of HTPAP, which might be a novel determinant and target for HCC prognosis.