热休克蛋白70 C末端反应蛋白对人小细胞肺癌细胞株的作用

Effect of C-terminus of Hsp70-interacting Protein on Human Small Cell Lung Cancer Cell Lines

目的探讨热休克蛋白70C末端反应蛋白(C-terminus of hsp70-interacting protein,CHIP)过表达对MET以及SCLC细胞株凋亡以及侵袭功能的影响。方法体外培养SCLC细胞株,免疫印迹法检测SCLC细胞系中CHIP,MET的表达关系。用pcDNA3-CHIP(+)和pcDNA3(-)构建的NCI-H69细胞,检测ERK,FAK,pERK1/2,Akt,pFAK,GFP,Myc和paxillin的表达,评价CHIP和MET蛋白的表达。用GFP标记的CHIP和Myc标记的MET表达载体共转染NCI-H69细胞,免疫沉淀方法检测CHIP与MET的相互作用。最后对CHIP转染的细胞行细胞活力、细胞凋亡和细胞侵袭试验。结果CHIP与MET在SCLC细胞系中的表达呈反比关系。转染pcDNA3-CHIP(+)后的细胞CHIP蛋白表达升高,同时MET蛋白表达降低。CHIP过表达的肿瘤细胞中的paxillin和FAK活化程度降低。CHIP过表达SCLC细胞株的侵袭能力下降,细胞生长活力明显下降,凋亡水平增加。转染阴性对照载体(-)或CHIP表达载体(+)的NCI-H69细胞中CHIP过表达后MET的聚泛素化水平升高。结论CHIP抑制MET依赖性信号,调节MET介导的SCLC运动、凋亡和侵袭。此外,CHIP通过在SCLC细胞系中的过表达,可明显减少细胞侵袭和生长,同时促进细胞凋亡。

Objective To investigate the effect the overexpression of C-terminus of hsp70-interacting protein(CHIP)on the apoptosis and invasion of MET and SCLC cells lines.Methods SCLC cell lines were cultured in vitro,and the expression of CHIP and MET in SCLC cell lines were detected by Western blot.NCI-H69 cells constructed by transient transfection of pcDNA3-CHIP(+)and pcDNA3(-)were used to detect the expression of ERK,FAK,pERK1/2,Akt,pFAK,GFP,Myc,and paxillin.The expression of CHIP and MET proteins was evaluated.Meanwhile,the expression vectors of GFP-labeled CHIP and Myc-labeled MET were co-transfected into NCI-H69 cells,and the interaction between CHIP and MET was detected by immunoprecipitation.Finally,the cell viability,apoptosis and invasion of the cells transfected with CHIP were tested.Results There was an inverse relationship between CHIP and MET expression in SCLC cell lines.After transfection with pcDNA3-CHIP(+),the expression of CHIP proteins increased,while that of MET proteins decreased.The activation of paxillin and FAK decreased in CHIP overexpressed tumor cells.The overexpression of CHIP in SCLC cells showed decreased invasion,significantly decreased cell growth and increased apoptosis.In NCI-H69 cells transfected with negative control vector(-)or chip expression vector(+),the polyubiquitination level of MET increased after CHIP overexpression.Conclusion CHIP inhibits dependent signaling of MET and regulates the movement,apoptosis and invasion of SCLC mediated by MET.In addition,CHIP can significantly reduce cell invasion and growth and promote cell apoptosis through its overexpression in SCLC cell lines.