目的证实接合物蛋白CrkⅠ在卵巢癌恶性潜能中的作用。方法采用Western blot法检测卵巢癌组织、卵巢良性肿瘤组织、正常卵巢组织中Crk和Dock180蛋白的表达;用免疫沉淀法检测3种卵巢癌细胞株中Crk与Dock180蛋白的内源性结合;用小干扰RNA敲低SKOV3细胞中内源性的Crk,检测Crk表达缺失性细胞Crk蛋白表达水平、Rac1酶活性和侵袭力的变化。结果 Dock180与CrkⅠ的表达强度呈现明显的一致性,卵巢癌组织中二者的表达均显著高于卵巢良性肿瘤组织和正常卵巢组织(P〈0.05);而在卵巢良性肿瘤组织与正常卵巢组织之间未发现明显差异(P〉0.05)。3个卵巢癌细胞株中Dock180主要表现出与CrkⅠ的内源性结合。和对照相比,敲低Crk基因的细胞表现出侵袭能力和Rac1酶活性明显下降(P〈0.05),而敲低CrkⅠ的细胞和同时敲低CrkⅠ和CrkⅡ的细胞相比无明显差异(P〉0.05)。结论卵巢癌的恶性生物学行为与Crk/Dock180/Rac1信号通路相关,其中CrkⅠ在卵巢癌中扮演着主要角色。
Objective To explore the role of adaptor protein CrkⅠ in malignant potential of human ovarian cancer.Methods Crk and Dock180 expression were detected by Western blotting in ovarian cancer tissues(EOC,n=28),benign ovarian tumors(BOT,n=13) and normal ovary tissues(Normal,n=10).Co-precipitation was performed to evaluate the in vivo protein-protein interaction of Dock180 and Crk in 3 different ovarian cancer cell lines(SKOV3,MCAS and RMUG-L cells).The expression of Crk in SKOV3 cells were silenced by using siRNA interference,and then Rac1 activity and cell invasion in the transfected cells were observed.Results The intensity of CrkⅠ and Dock180 expression was consistent with each other in EOC tissues.Both were observed to be significantly higher in EOC than those in the BOT and normal ovary tissues(P0.05).No significant difference was found between BOT and Normal group either for CrkⅠor Dock180 expression(P0.05).In consistent with this result,Dock180 preferred to combine with CrkⅠ rather than with CrkⅡ in all 3 ovarian cancer cell lines.Furthermore,Crk knockdown cells presented with sustainable CrkⅠ expression depletion,significantly decreased Rac1 activity and cell invasion.Conclusion Crk might be involved in malignant potential of human EOC mainly through CrkⅠ/Dock180/Rac1 pathway.