摘要
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。
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 knock-down 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.
出处
《细胞生物学杂志》
CSCD
2007年第6期889-894,共6页
Chinese Journal of Cell Biology
基金
辽宁省自然科学基金资助项目(No.20061074)~~
关键词
肝细胞癌
侵袭和转移
黏着斑激酶
RHOA
上皮-间叶转化
hepatocellular carcinoma
invasion and metastasis
focal adhesion kinase
RhoA
epithelialmesenchymal transition
