特异性下调葡萄糖调节蛋白78表达对肝细胞癌侵袭和转移能力的影响

为了研究特异性下调葡萄糖调节蛋白(Grp)78对肝细胞癌侵袭和转移能力的影响。通过小干扰RNA(siRNA)技术特异性下调人肝细胞癌细胞株BEL7402中Grp78的表达,并应用Transwell法和划痕法对肝细胞癌侵袭、转移能力的改变进行分析,应用免疫沉淀技术和GST-pulldown技术分别对黏着斑激酶(FAK)的磷酸化水平和小GTPase RhoA的活性进行研究,应用免疫印迹技术检测E-钙黏着蛋白、N-钙黏着蛋白和波形蛋白的表达。结果发现,Transwell实验和划痕实验结果显示特异性下调Grp78表达可以抑制肝细胞癌的侵袭和转移,免疫沉淀结果显示特异性下调Grp78表达可以降低FAK的磷酸化水平,GST-pulldown实验结果表明特异性下调Grp78表达可以上调RhoA的活性。免疫印迹实验结果表明特异性下调Grp78可以下调N-钙黏着蛋白、波形蛋白的表达,上调E-钙黏着蛋白的表达。结果表明特异性下调Grp78在体外可以抑制肝细胞癌的侵袭和转移,这种抑制作用是通过FAK脱磷酸化和抑制肿瘤的上皮-间叶转化实现的。

The Effects of Specific Down-regulation of Grp78 by siRNA on Invasion and Metastasis of Hepatocellular Carcinoma

To investigate the roles of glucose-regulated protein 78 (Grp78) in invasion and metastasis of hepatocellular carcinoma (HCC), we down-regulated the expression of Grp78 by small interfering RNA (siRNA) in human HCC cell lines BEL 7402, the invasion and metastasis behaviors of HCC in Grp78 knock-down cells was analyzed by Transwell assay and wound healing assay. Focal adhesion kinase (FAK) phosphorylation was analyzed by immunoprecipitation. The activity of RhoA was determined by GST-pulldown assay. The expression status of E-cadherin, N-cadherin and vimentin was examined by Western blot. We found that the invasion and metastasis capabilities of HCC in Grp78 knock-down cells were significantly decreased compared with parental cells. Phosphorylation of FAK was significantly inhibited and RhoA activity was promoted in Grp78 knock-down cells. The expression levels of N-cadherin, vimentin were significantly lower, while E-cadherin was higher in Grp78 knockdown cells than in normal cells. We concluded that Grp78 silencing may inhibit the invasion and metastasis of HCC, These data suggest that Grp78 may be a potential target for HCC gene therapy.