鑑定基質金屬蛋白酶-7 變異體為肝硬化之新危險因子

Abstract

Hepatocellular carcinoma (HCC) usually occurs in cirrhotic liver, but 10-30% of HCC occur in non-cirrhotic liver. Several independent risk factors were associated with an increased rate of fibrosis progression: chronic hepatitis C virus infection, male gender, increased body mass index and alcohol abuse. However, these factors have not been useful in accurately predicting which patients will progress to cirrhosis. A generally held hypothesis is that host genetic factors may explain this different individual behavior. To identify genetic determinants of susceptibility to cirrhosis would assist in predicting individual risks of disease progression and would help to clarify pathophysiological mechanisms of liver cirrhosis. Fibrosis and cirrhosis are characterized by progressive accumulation of extracellular matrix that follows chronic liver injuries. In the extracellular space, the constant turnover of liver matrix is regulated by a class of enzyme called matrix metalloproteinase (MMP). To assess whether genetic variations in matrix turnover influence the diversity of liver cirrhosis, a case-control study of 320 HCC patients with or without cirrhosis was conducted. Ten single nucleotide polymorphism (SNP) markers from 4 candidate genes were selected and genotyped by Beckman SNPstream genotyping platform. Among these genes, a nonsynonymous single nucleotide polymorphism which generates the variation of Asp-137 in MMP-7 gene was found to be strongly associated with the development of liver cirrhosis. Hepatic stellate cell lines HSC-T6-MMP7(Gly) and HSC-T6-MMP7(Asp) that stably express MMP-7(Gly-137) and MMP-7(Asp-137), respectively, were generated. Different from MMP-7(Gly-137) that mostly secreted into cultured medium, MMP-7(Asp-137) distributed to the cell surface where it exerts its proteolytic activity on pericellular substrates. In addition, enzymatic activities and immunoreactive active forms of MMP-7 were detected in Triton-resistant fraction. Functional analysis demonstrated an increased ability of the MMP-7(Asp-137) variant to interact with cell surface molecule CD151. Furthermore, cell motility was enhanced with the expression of the MMP-7(Asp-137). Pretreatment with neutralizing antibody to MMP-7 specifically blocked the cell migration. These results suggested that not only the membrane anchorage but also its activity is important for promoting cell migration. To investigate the molecular mechanisms by which MMP-7(Asp-137) variant enhances cell migration, substrates that involve cell-cell interaction were analyzed. Shedding of E-cadherin was increased in cells expressing MMP-7(Asp-137) variant in comparison with cells expressing MMP-7(Gly-137). These results demonstrated that the MMP-7(Asp-137) variant confers a gain-of-function phenotype for MMP-7. The present study not only identified a genetic association between MMP-7(Asp-137) variant and liver cirrhosis but also uncovered the effects of SNP on functional phenotypes. These findings support a role for MMP-7 G137D polymorphism in the pathogenesis of liver cirrhosis, suggesting a potential marker for predicting cirrhosis progression and prognosis.