CD151及其整合素突变体对肝癌细胞侵袭能力的影响及机制

Effects and mechanism of CD151 and its integrin mutant on invasion ability of liver cancer cells

目的探讨CD151及其整合素结合突变体(CD151-MUT)对肝癌细胞侵袭能力的影响,并探讨其与FAK-Src-paxillin信号通路的关系。方法取人肝癌细胞株HepG2,随机分为对照组、CD151组和CD151-MUT组。对照组加入生理盐水,CD151组和CD151-mut组分别转染pAAV-CD151质粒、pAAV-CD151-AAA质粒,孵育4 h。继续培养24 h后收集细胞,采用Transwell小室实验观察各组细胞侵袭能力,Western blotting法检测各组细胞CD151及FAK-Src-paxillin通路活性,免疫共沉淀法检测CD151、CD151-mut与整合素α3、α6、β1的结合情况。结果CD151组Transwell小室实验穿膜细胞数较对照组增加,CD151-mut组穿膜细胞数较CD151组减少(P均<0.05)。CD151组、CD151-mut组CD151蛋白表达均较对照组表达增加(P均<0.05),CD151组与CD151-mut组比较差异无统计学意义。CD151组p-FAK、p-Src、p-paxillin蛋白表达水平较对照组升高,CD151-mut组p-FAK、p-Src、p-paxillin蛋白表达水平较CD151组降低。免疫共沉淀结果显示,CD151与整合素α3、α6和β1结合形成复合体,CD151组结合整合素α3、α6、β1水平高于对照组(P均<0.05),而CD151-mut组结合整合素α3、α6、β1水平低于CD151组(P均<0.01)。结论CD151高表达可促进肝癌细胞的侵袭,破坏CD151和整合素复合体的形成则细胞的侵袭能力减弱;该机制与调控FAK-Src-paxillin信号通路蛋白的活性有关。

Objective To investigate the effects of CD151 and its integrin binding mutant(CD151-mut)on the invasion of hepatoma cells and its relationship with FAK-Src-paxillin signaling pathway.Methods HepG2 cells were randomly divided into the control group,CD151 group and CD151-mut group.Cells in the control group were added with normal saline,while cell in the CD151 group and CD151-mut group were transfected with pAAV-CD151 and pAAV-CD151-AAA plasmids,respectively,and then were incubated for 4 hours.After 24 hours of continuous culture,the cells were collected,and the invasive ability in each group was observed by Transwell chamber experiment.The activities of CD151 and FAK-Src-paxillin pathway were detected by Western blotting,and the CD151,CD151-mut and integrinα3,α6,β1 were detected by immunocoprecipitation.Results The number of transmembrane cells in the CD151 group was higher than that in the control group,and that in the CD151-mut group was lower than that in the CD151 group(both P<0.05).The expression of CD151 protein in the CD151 group and CD151-mut group was higher than that in the control group(P<0.05).The expression levels of p-FAK,p-Src and p-paxillin in the CD151 group were higher than those in the control group,while the expression levels of p-FAK,p-Src,and p-paxillin in the CD151-mut group was lower than those in the CD151 group.The results of immunocoprecipitation showed that CD151 combined with integrinα3,α6 andβ1 to form complexes.The levels of integrinα3,α6 andβ1 in the CD151 group were higher than those in the control group(all P<0.05),while the levels of integrinα3,α6 andβ1 in the CD151-mut group were lower than those in the CD151 group(all P<0.01).Conclusion The overexpression of CD151 can promote the invasion of hepatoma cells,and the destruction of CD151 and integrin complex will weaken the invasion of hepatoma cells,which is related to the regulation of the activity of FAK-Src-paxillin signaling pathway protein.