Role Of Wnt Inhibitory Factor-1 In Hepatocellular Carcinoma Cells

Although HCC(hepatocellular carcinoma) is one of the most common cancers worldwide and a major cause of death in many countries,especially in China,the molecular changes and mechanisms that regulate its development and progression remain unclear. Recently,abnormal activation of the WNT pathway has been found to be involved in the carcinogenesis of several human cancers including HCC.The WNT signaling pathway is essential for a wide array of developmental and physiological processes.Wnts are extracellular ligands that bind to frizzled(Fz) receptors at the membrane,canonically inducingβ-catenin nuclear translocation and activation,where it associates with the T-cell factor(TCF) family of transcription factors.Many articles report that aberrant activation of the WNT signaling pathway exists in many HCC cell lines, however,mutation of APC orβ-catenin is not common in HCC cell lines.Our results and previous reports suggest that other mechanisms may be involved in the activation of the WNT signaling in HCC.WNT inhibitory factor-1(WIF-1) is a secreted protein that antagonizes WNT signaling and has important implications for carcinogenesis.Low expression and promoter hypermethylation of WIF-1 genes is common in human cancers.However,the role of WIF-1 in HCC is not clear.We found that the level of WIF-1 expression is low in HCC cell lines compared with normal tissue,which is associated with promoter hypermethylation.CpG methylation-dependent silencing of WIF-1 was observed among HCC cell lines(5/8;63.5%) by methylation-specific PCR and direct sequencing.WIF-1 expression in SMMC-7721, HepG2 and BEL-7402 could be rapidly restored by treatment of DNA methyltransferase inhibitor 5-aza-2-deoxycytidine.The above results further indicated the relationship between the methylation status on WIF-1 promoter and the expression of the gene in HCC cell lines.To explore whether the down-regulation of WIF-1 contributes to hepatocarcinogenesis, we transiently transfected plasmid pcDNA3.1-WlF-1 into SMMC-7721,a HCC cell line without the significant expression of endogenous WIF-1.The results showed that exogenous WIF-1 could significantly inhibit the cell growth of SMMC-7721 cells compared with the vector control,suggested that WIF-1 as an antagonist of WNT pathway play important roles in suppressing the cell growth of HCC cells.To further conform the effect of WIF-1 on the cell growth of HCC cells,the colony formation assay was performed.The cells were incubated for 21 days in G418.The colony formation of SMMC-7721 cells tranfected with plasmid pcDNA3.1-WIF-1 was dramatically decreased.Our result suggests that WIF-1 is a potent inhibitor for negatively regulating the cell growth through the opposed effect of WNT-β-catenin pathway,which is well-known as an important contributor to the oncogenesis of HCC.Taken together,our studies implicate that WIF-1 silencing is an important mechanism for the constitutively activated WNT signaling in HCC.WIF-1 may be a potential candidate for treatment of HCC by inhibition of the WNT pathway.