Primary hepatocellular carcinoma (HCC) is one of the most common malignancies all over the world. However, the mechanism of HCC initiation and development remains unclear. In our previous work, high-throughput microarray assay in HCC samples followed by bioinformatic analysis suggested that Metallothionein1G (MT1G) might be one of the key factors in HCC. In this study, we aim to clarify the biological function of MT1G and validate its potential to be utilised as a biomarker in HCC.Methods
We detected the MT1G expression in paired HCC samples and cell lines by both RT-qPCR and Western blot. MSP (Methylation specific PCR) and BGS (Bisulfite genomic sequencing) were performed to evaluate methylation status of MT1G in HCC. TCGA (The Cancer Genome Altas) data analysis was used to validate the results from our samples. The functional significance of MT1G was investigated by overexpression or knockdown in vivo and in vitro. Kaplan-Meier survival analysis was performed using TCGA data to estimate the clinical value of MT1G expression and methylation status in HCC.Results
MT1G was inactivated in 4 of 6 HCC cell lines. The expression of MT1G was downregulated in cancer tissues compared with the adjacent non-tumour tissues (p<0.001). The gene expression of MT1G was closely correlated to the promoter methylation status. The MT1G expression in silenced HCC cell lines could be restored by demethylation agent. Ectopic re-expression of MT1G by stable transfection in SMMC7721 and Hep3B cells inhibited colony formation (p<0.001), suppressed cell motility and invasiveness (p<0.05), accompanying with up-regulation of E-cadherin; and down-regulation of PCNA, MMP2, MMP13 and Vimentin. Xenograft tumour assay in nude mice also revealed that MT1G could markedly decrease tumour weights and volumes in vivo (p<0.001). Survival analysis revealed that hypermethylation of MT1G predicts good outcomes of HCC patients.Conclusions
Our results demonstrate that MT1G promoter methylation directly mediates the transcription down-regulation and commonly occurs in HCC. MT1G gene can act as a functional tumour suppressor in liver carcinogenesis by playing an important role in the depression of cell proliferation, migration and invasion.