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中华肺部疾病杂志(电子版)

中华肺部疾病杂志(电子版) ›› 2019, Vol. 12 ›› Issue (06) : 697 -701. doi: 10.3877/cma.j.issn.1674-6902.2019.06.006

论著

YAP调控肺癌A549/DDP细胞顺铂耐药性的机制分析
肖丹1, 杜琴1, 贺斌峰2, 郭晓兰1,†()   
  1. 1. 637007　南充，川北医学院转化医学研究中心
    2. 400037　重庆，陆军军医大学（第三军医大学）第二附属医院呼吸研究所
  • 收稿日期:2019-05-17 出版日期:2019-12-20
  • 通信作者: 郭晓兰
  • 基金资助:
    四川省卫计委科研课题(16PJ131)

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

Dan Xiao1, Qin Du1, Binfeng He2, Xiaolan Guo1,†()   

  1. 1. Research Center of Translational Medicine, North Sichuan Medical College, Nanchong 637007, Sichuan Province, China
    2. Institute of Respiratory Diseases, Xinqiao Hospital, Army Military Medical University, Chongqing 400037, China
  • Received:2019-05-17 Published:2019-12-20
  • Corresponding author: Xiaolan Guo
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肖丹, 杜琴, 贺斌峰, 郭晓兰. YAP调控肺癌A549/DDP细胞顺铂耐药性的机制分析[J]. 中华肺部疾病杂志(电子版), 2019, 12(06): 697-701.

Dan Xiao, Qin Du, Binfeng He, Xiaolan Guo. Effect and mechanism of Yse-associated protein controlling drug resistance of cisplatin in A549/DDP cells of patients with lung cancer[J]. Chinese Journal of Lung Diseases(Electronic Edition), 2019, 12(06): 697-701.

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(IC50)。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。

关键词: 非小细胞肺癌, 维替泊芬, YAP, 顺铂耐药
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 (IC50) 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.

Key words: Non-small cell lung cancer, Verteporfin, YAP, Cisplatin resistance
图1 A549/DDP抵抗顺铂的杀伤作用；注：A：MTS顺铂处理A549以及A549/DDP细胞的细胞活力曲线；B：A549以及A549/DDP细胞的顺铂IC50；注：*与A549细胞相比，P<0.05
图2 检测YAP在A549/DDP中的表达；注：A：qPCR检测YAP mRNA在A549以及A549/DDP细胞中的表达；*，与A549细胞相比，*P<0.05；B：Western blot检测YAP蛋白在A549以及A549/DDP细胞中的表达
图3 VP增强A549/DDP细胞对顺铂敏感性；注：A：Western blot检测VP处理后A549/DDP细胞中YAP蛋白表达；B：经药物处理后A549/DDP细胞的细胞活力；注：*，与DMSO组相比，*P<0.05；^，与DDP组相比，^P<0.05
图4 qPCR检测A549/DDP细胞经VP处理后干细胞标志物mRNA的表达变化；注：*，与DMSO组相比，*P <0.05
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