RACK1通过影响MCM7磷酸化促进肺癌细胞的增殖

目的:研究蛋白激酶C受体1(receptor for activated C kinase 1,RACK1)基因沉默或过表达对大细胞肺癌细胞系H460及肺腺癌细胞系A549细胞增殖的影响,并探讨其可能的作用机制.方法:采用脂质体转染法分别将针对RACK1基因的RACK1 siRNA和重组质粒pCMV-sport6-RACK1转入H460和A549细胞中.采用MTT法和集落形成实验分析RACK1基因对细胞增殖的影响,FCM检测其对细胞周期的影响；采用酵母双杂交法、免疫共沉淀法、激光共聚焦显微术及磷酸化蛋白免疫共沉淀法检测RACK1表达与肺癌细胞增殖之间的关系.结果:转染RACK1 siRNA后,H460和A549细胞中RACK1蛋白的表达量明显下调,细胞的增殖能力和集落生成数明显降低,S期细胞所占比例明显下降(P＜0.01)；转染pCMV-sport6-RACK1后,H460和A549细胞中RACK1蛋白的表达量明显上调,细胞的增殖能力增强,倍增时间增加,集落生成数明显增多,S期细胞所占比例明显升高(P＜0.01).采用酵母双杂交法和免疫共沉淀法检测发现,RACK1与MCM7存在直接作用.RACK1进入细胞核内,与微小染色体维持蛋白7 (minichromosome maintenance protein 7,MCM7)特异性结合,促进MCM7蛋白磷酸化.结论:RACK1通过直接与MCM7结合促进MCM7蛋白磷酸化途径,继而促进肺癌细胞增殖.

RACK1 promotes the proliferation of lung cancer cells by targeting phosphorylation of MCM7 protein

Objective:To investigate the effects of gene silencing and overexpression of RACK1(receptor for activated C kinase 1) on the proliferation of large-cell lung cancer H460 cells and lung adenocarcinoma A549 cells,and to explore the possible mechanism.Methods:The RACK1 siRNA(small interfering RNA) targeting RACK1 gene and recombinant vector pCMV-sport6-RACK1 were transfected into both of H460 cells and A549 cells,respectively.MTT method and colony formation assay were used to detect the effect of RACK1 gene expression on the proliferation of lung cancer cells.Flow cytometry was used to examine the change of cell cycle.The association and interaction of RACK1 gene expression with the proliferation of lung cancer cells were analyzed by yeast two-hybrid system,co-immunoprecipitation,laser scanning confocal microscopy and co-immunoprecipitation of phospho-proteins.Results:The expression levels of RACK1 protein in the H460 cells and A549 cells were both decreased after transfection with RACK1 siRNA,and the abilities of proliferation and colony-formation were also weakened.The proportion of lung cancer cells arrested at phase S was significantly declined(P<0.01).Meanwhile,the expression level of RACK1 protein was increased after transfection with pCMV-sport6-RACK1,and the abilities of proliferation and colony-formation of lung cancer cells were both strengthened with a prolonged doubling time.The proportion of lung cancer cells arrested at phase S was significantly increased(P<0.01).The results of yeast two-hybrid system and co-immunoprecipitation revealed that RACK1 could directly interact with MCM7(minichromosome maintenance protein 7).The phosphorylation of MCM7 protein was strengthened through binding to RACK1 which translocated into the cell nucleus.Conclusion:RACK1 promotes the proliferation of lung cancer cells through activating the phosphorylation of MCM7 binding to RACK1.