鑑定基質金屬蛋白酶-7 變異體為肝硬化之新危險因子

Abstract

肝炎、肝硬化乃至肝癌是我們常見的肝病發展三部曲，然而臨床上發現，10~30%的肝癌患者是在不經過肝硬化的病程下直接發生。研究指出，伴隨肝硬化發生的肝癌患者預後較差，復發機率較沒有硬化者來的高。許多流行病學研究在尋找決定肝硬化發生的危險因子，結果顯示肝硬化的發生與酗酒、性別是男性、C型肝炎病毒感染等危險因子有關，但是我們發現即使暴露在相同的危險因子下，個體間仍有不同的病程發展。因此，我們推測個體的遺傳背景可能也是導致疾病多樣性的原因之一。為了測試這個假設，我們選擇四個與纖維化相關的基因（MMP-7、MMP-13、PAI-1以及CFTR），針對其中十個單點核苷酸多型性 (SNP) 來進行基因型分析。一個位在MMP-7基因上，使第137胺基酸從glycine (Gly，G) 轉變成aspartic acid (Asp，D) 的SNP和肝硬化的發生有統計上的關聯性。從功能性試驗中發現，不同於MMP-7(Gly-137) 主要是分泌到細胞外，MMP-7(Asp-137) 變異體反而是座落在細胞膜上為主。分泌型的MMP-7可藉由與細胞膜上的分子結合而座落於細胞表面，此過程將有助於MMP-7的活化。因此，進一步比較Gly-137與Asp-137兩種型式的MMP-7和細胞膜上分子的結合能力，結果發現MMP-7(Asp-137) 變異體與CD151的結合能力優於MMP-7(Gly-137)，這樣的特性使得Asp-137型式的MMP-7增加機會停留於細胞表面，繼而增加活化機會。 在評估細胞移動能力的試驗中，結果發現表現Asp-137型式的MMP-7會加速肝臟星狀細胞的移動。肝臟星狀細胞的移動能力越好，可加速其釋放纖維化蛋白質來修復傷口的過程。因此，對肝硬化的病程發展是一個正向調控機制。已知MMP-7的基因功能是透過破壞細胞與細胞以及細胞與間質之間的結合，來進行細胞移動的調控，在表現MMP-7(Asp-137) 變異體的細胞中，我們觀察到全長E-cadherin分子的切割，比起表現MMP-7(Gly-137)的細胞明顯，推測此為MMP-7(Asp-137)變異體加速細胞移動的部分機制。 本研究發現MMP-7(G137D) polymorphism是全新的肝硬化危險因子，並闡明此因子促進肝硬化的功能機轉。透過這個研究，除了有助於肝硬化致病機轉的了解之外，並且可作為個體預測疾病感受性的標誌，有利於預防醫學的推動。

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.