沉默Rheb对DDP耐药非小细胞肺癌细胞活力和凋亡的影响及机制研究

Effect and mechanism of si-Rheb on viability and apoptosis of DDP-resistant nun-small cell lung cancer cells

目的:研究沉默Rheb对DDP耐药非小细胞肺癌(NSCLC)细胞活力和凋亡的影响,并探索其作用机制。方法:体外培养非小细胞肺腺癌细胞A549和顺铂耐药的A549细胞(A549/DDP)。转染Rheb-siRNA(si-Rheb)至A549和A549/DDP细胞。采用CCK-8法、Annexin-V/PI双染,qPCR和Western blotting方法检测si-Rheb对细胞活力和凋亡的影响。结果:Rheb在A549/DDP细胞中高表达(P<0.05)。转染si-Rheb可有效抑制A549和A549/DDP细胞中Rheb的表达,显著降低A549和A549/DDP细胞活力,诱导细胞凋亡,促进A549/DDP对DDP的敏感性(P<0.05)。此外,转染si-Rheb显著影响了凋亡相关蛋白,如细胞色素C、RARP和Caspase-3表达,并抑制了mTOR-S6信号通路的过度激活(P<0.05)。结论:Rheb通过mTOR-S6信号通路调控DDP耐药A549细胞对DDP的敏感性。

Objective:To explore the effects of Rheb knockdown on viability and apoptosis of DDP-resistant non-small cell lung cancer(NSCLC)cells and its possible mechanisms.Methods:Non-small cell lung adenocarcinoma cell A549 and DDP-resistant A549(A549/DDP)cells were cultured in vitro.Transfected Rheb-siRNA(si-Rheb)into A549 and A549/DDP cells.CCK-8 assay,Annexin-V/PI double stain kit,qPCR and Western blotting were used to investigate the effects of Rheb siRNA(si-Rheb)on viability and apoptosis of cells.Results:Rheb was upregulated in A549/DDP cells(P<0.05).Transfection with si-Rheb into A549 and A549/DDP cells significantly inhibited expression of Rheb,significantly suppressed cell viability,induced cell apoptosis,and promoted sensibility of A549/DDP to DDP(P<0.05).In addition,si-Rheb markedly effected expressions of apoptosis related proteins,such as Cytochrome-C,RARP and Caspase-3,and suppressed overactivated of mTOR-S6 signal pathway(P<0.05).Conclusion:Rheb promoted the sensitivity of A549/DDP cells to DDP may through regulating the mTOR-S6 signal pathway.