沉默YAP逆转肺癌PC9细胞多柔比星耐药性及其机制研究

背景与目的：耐药性是导致肺癌患者化疗失败的主要原因。探讨YAP对人肺癌PC9细胞多柔比星耐药的逆转作用及其机制。方法：利用体外筛选方法从多柔比星敏感性肺癌细胞系PC9获得耐药细胞克隆，并检测YAP的表达水平；利用shRNA沉默细胞中YAP的表达，应用MTS法检测肿瘤细胞药物敏感性，流式细胞术检测细胞周期、凋亡及对Rh-123的吸收能力，蛋白质]印迹法(Western blot)和实时定量聚合酶链式反应(quantitative real-time polymerase chain reaction，QRT-PCR)技术检测ABCB1、ABCC1、p53、Runx2、ITGB2和ErbB4的表达水平及丝氨酸/苏氨酸蛋白激酶(serine/threonine kinase，AKT)的磷酸化水平变化。结果：经体外诱导获得多柔比星耐药细胞克隆PC9/Adr，且YAP蛋白在其中高表达，利用shRNA得到不同YAP沉默程度的PC9/Adr。YAP沉默后，细胞生长速度降低，细胞对多柔比星的敏感性显著增加，细胞周期被阻滞在G0/G1期，多柔比星诱导的细胞凋亡增多，细胞吸收Rh-123也增多，并与YAP的沉默程度呈正相关。Western blot和QRT-PCR结果显示，YAP沉默后，ABCB1、ABCC1、Runx2、ITGB2和ErbB4蛋白表达下调，而p53的表达上调，AKT的磷酸化水平则下降。结论：YAP过表达与PC9/Adr的耐药性相关，沉默YAP可恢复PC9/Adr对多柔比星的敏感性。这一作用与调节耐药相关基因的表达、促进细胞凋亡有关。

YAP silencing reverses doxorubicin resistance in lung cancer cell line PC9 and its mechanism

Background and purpose:Drug resistance is a major cause of failure in lung cancer chemotherapy. This study aimed to investigate the effect of YAP on doxorubicin resistance in lung cancer and its underlying mechanism. Methods:Doxorubicin resistant lung cancer cell clones were established from parental sensitive cancer PC9 cell line via in vitroinduction, and the expression of YAP was analyzed. YAP was down-regulated via shRNA to different levels. MTS assay was employed to determine cell proliferation and drug sensitivity. Flow cytometry was used to determine cell cycle distribution, apoptosis and uptake of Rh-123. Western blot and quantitative real-time polymerase chain reaction (QRT-PCR) assay were used to determine the expression of ABCB1, ABCC1, p53, Runx2, ITGB2 and ErbB4. The phosphory-lation of serine/threonine kinase (AKT) was determined as well.Results:Doxorubicin resistant PC9/Adr cell clone was obtained with over-expressed YAP. Expression of YAP in PC9/Adr cells was down-regulated to different levels via shR-NA. After YAP silencing, cell proliferation was reduced, while sensitivity to doxorubicin was increased. The cell cycle was significantly halted by G0/G1 phase. Doxorubicin induced-apoptotic rate and cellular uptake of Rh-123 were increased,with positive correlation to YAP silencing level. Western blot and QRT-PCR results showed that after YAP silencing, ABCB1, ABCC1, Runx2, ITGB2, and ErbB4 proteins were down-regulated, while the expression of p53 was up-regulated. Phosphorylation of AKT was inhibited as well.Conclusion:Over-expression of YAP is involved in doxorubicin resistance in PC9/Adr cell line. Silencing of YAP could restore doxorubicin sensitivity. The mechanism involves regulation of drug resistance-related genes and promotion of apoptosis.