Hepatocellular carcinoma (HCC) remains a major cause of cancer-related death worldwide. Metallothioneins (MTs) are low-molecular weight, cysteine-rich proteins, and classified into multiple classes. One of the MT species, MT1H, is down regulated in HCC. However, the roles of MT1H in HCC are not fully understood.
The expression of MT1H in large datasets derived from TCGA databases. Hepatoma HepG2 and Hep3B cells were transfected with full length Flag-tagged MT1H. The cell growth curved was obtained through MTT assay. Invasiveness and migration ability of hepatoma cells was studied through the matrigel-coated transwell assay. HepG2-Vector and HepG2-MT1H cells were subcutaneously injected into nude mice for assessing in vivo tumorigenicity of MT1H over-expression.
The results from TCGA RNASeq2 data showed that MT1H is significantly down regulated in most HCC analyzed. MT1H expression level was found to be markedly decreased in all HCC tumors. Hepatoma HepG2 and Hep3B cells were transfected with full length Flag-tagged MT1H. Enforced expression of MT1H in HepG2 and Hep3B cells led to a more than 50% decrease in colony numbers as compared with control cells. The DNA synthesis capability was significantly decreased in MT1H-overexpressed hepatoma cells. Ectopic overexpression of MT1H significantly impaired the migration capability of hepatoma cells. We found that Wnt/ß-catenin pathway related genes were significantly enriched in the HCC patients with low MT1H expression. Consistently, ectopic over-expression of MT1H in HCC cells significantly suppressed the mRNA level of ß-catenin signaling downstream targets (c-Myc, MMP7, LEF1, and TCF4) . As expected, Wnt/ß-catenin nuclear translocation was significantly attenuated in MT1H over-expressed HCC cells. To assess the effect of MT1H over-expression on tumorigenicity in vivo, we subcutaneously injected nude mice with HepG2-Vector and HepG2-MT1H cells. The tumors formed by HepG2-MT1H cells were significantly smaller than that of control cells. The average tumor weight of the MT1H- transfected cells inoculated mice was significantly decreased at day 30.
Our findings suggest that MT1H functions as tumor suppressor in HCC mediated by Wnt/ß-catenin signaling pathway.
Legal entity responsible for the study
Zhejiang Provincial Natural Science Fund (grant no. LY13H160007) and the Zhejiang Medicines and Health Science and Technology Project (grant no. 201348801).
All authors have declared no conflicts of interest.