YAP调控肺癌A549/DDP细胞顺铂耐药性的机制分析

Effect and mechanism of Yse-associated protein controlling drug resistance of cisplatin in A549/DDP cells of patients with lung cancer

目的探讨Yse相关蛋白(Yes-associated protein,YAP)对A549/DDP细胞顺铂耐药性的影响及其机制。方法采用MTS检测顺铂对肺腺癌A549细胞以及肺腺癌耐顺铂A549/DDP细胞的50%抑制浓度(IC 50)。使用qPCR以及Western blot检测A549和A549/DDP细胞中YAP mRNA以及蛋白的表达。Western blot检测维替泊芬(Verteporfin,VP)处理A549/DDP后YAP蛋白表达变化。将A549/DDP细胞设置为DMSO对照组、顺铂组(DDP组)、维替泊芬组(VP组)、顺铂联合维替泊芬组(DDP+VP组),采用MTS检测0、24和48 h各处理组细胞活力的变化。采用qPCR检测VP处理A549/DDP后干细胞标志物ALDHA1、CD133、OCT4、NANOG、SOX2的mRNA表达变化。结果顺铂对A549和A549/DDP细胞的IC50分别为(4.07±0.03)μg/ml、(23.44±0.98)μg/ml,耐药指数为5.76。A549/DDP细胞中YAP显著高于A549细胞(P<0.05)。VP处理A549/DDP细胞后YAP蛋白表达水平较DMSO组明显降低。DDP+VP组较单独DDP组,其细胞活力在24 h和48 h均明显降低(P<0.05)。qPCR检测发现A549/DDP细胞经VP处理后,干细胞标志物ALDHA1、CD133、OCT4、NANOG、SOX2的mRNA均较DMSO组明显降低(P<0.05)。结论YAP可能参与了肺癌细胞的顺铂耐药。抑制YAP可通过抑制肿瘤干细胞特性,进而发挥逆转耐药的作用。

Objective To investigate the effect and mechanism of Yse-associated protein(YAP)on the drug resistance of cisplatin in the A549/DDP cells of the patients with lung cancer.Methods MTS was used to detect the 50%inhibitory concentration(IC 50)of cisplatin in the lung adenocarcinoma A549 cells and lung adenocarcinoma-resistant cisplatin A549/DDP cells.The expression levels of YAP mRNA and protein in the A549 and A549/DDP cells were detected by quantitative polymerase chain reaction(qPCR)and Western blotting.Western blotting was used to detect the expression level of YAP protein after A549/DDP cells receiving treatment of verteporfin(VP).The A549/DDP cells were designed as the DMSO control group,cisplatin group(DDP group),verteporfin group(VP group),and cisplatin combined with verteporfin group(DDP+VP group).And MTS was used to detect the cell viability in each treatment group immediately,24 h,and 48 h after treatment.The changes of mRNA expression of the stem cell markers ALDHA1,CD133,OCT4,NANOG and SOX2 after A549/DDP cells receiving treatment of VP were detected by qPCR.Results The IC50 values of cisplatin in the A549 and A549/DDP cells were(4.07±0.03)μg/ml and(23.44±0.98)μg/ml,respectively.YAP was significantly higher in the A549/DDP cells than in the A549 cells(P<0.05).The expression level of YAP protein in the VP-treated A549/DDP cells was significantly lower than that of the DMSO group.Compared with the DDP group,the DDP+VP group showed a significant decrease in the cell viability 24 h and 48 h after treatment(P<0.05).After treatment with VP,the mRNA expression levels of the stem cell markers ALDHA1,CD133,OCT4,NANOG and SOX2 were significantly lower than those of DMSO group(P<0.05).Conclusion YAP may be involved in the cisplatin resistance in the lung cancer cells.Inhibition of YAP can play a role in reversing drug resistance through inhibiting the characteristics of cancer stem cells.