摘要
目的探讨10号染色体同源缺失性磷酸酶-张力蛋白（PTEN）/蛋白激酶B（Akt）通路及泛素蛋白连接酶2（MDM2）基因在肺腺癌细胞耐药过程中的作用机制。方法构建肺腺癌耐药细胞A549/多西他赛（DTX）,实时定量聚合酶链反应检测A549细胞及A549/DTX细胞PTEN、Akt、MDM2基因表达差异;质粒转染干扰A549/DTX细胞PTEN、MDM2表达水平,3-（4，5-二甲基噻唑-2）-2，5-二苯基四氮唑嗅盐实验检测A549/DTX细胞对DTX半数抑制浓度（IC50）变化,实时定量聚合酶链反应及Westernblot检测Bcl-2、caspase-3表达变化;腺病毒转染构建过表达PTEN的A549/DTX细胞,行裸鼠荷瘤实验,分析DTX处理下A549/DTX移植瘤及A549/DTX-ad-PTEN移植瘤肿瘤体积、生长速度及MDM2、Bcl-2、caspase-3表达变化。结果构建的耐药株A549/DTX细胞DTX的IC50值较亲本株明显升高（P〈0.05）,并对顺铂、5-氟尿嘧啶出现交叉耐药;其PTEN基因表达较A549细胞明显降低,Akt、MDM2表达明显升高（P值均〈0.05）;PTEN表达水平与MDM2表达水平负相关,质粒转染诱导A549/DTX细胞PTEN过表达或MDM2沉默,A549/DTX细胞DTX的IC50值明显减小,对DTX敏感度增加,凋亡相关蛋白Bcl-2表达降低,caspase-3表达增加（P值均〈0.05）。裸鼠荷瘤实验提示PTEN过表达可诱导MDM2基因沉默,缩小移植瘤体积,减缓肿瘤生长速度,Bcl-2表达降低,caspase-3表达增加（P值均〈0.05）。结论PTEN/Akt信号通路过度活化,诱导MDM2高表达,可诱导肺腺癌细胞增殖增加、凋亡减少,促进肺腺癌细胞耐药。外源性干预降低MDM2表达水平,可能成为逆转肺腺癌细胞耐药的靶点之一。
Objective To investigate the mechanism of phosphatase and tensin homologue deleted on chromosome ten （PTEN）/protein kinase B （Akt） pathway and murine double mimute 2 （MDM2） gene expression in drug resistance of lung adenocarcinoma cells. Methods Lung adenocardnoma Docetaxel （DTX）-resistant cell A549/DTX was constructed. The expressions of PTEN, Akt and MDM2 in A549 and A549/DTX cells were detected by real-time polymerase chain reaction. RNA interference specific plasmid vector for MDM2 （siMDM2） and negative control （siNC） were transfected into test cell line, and then half maximal inhibitory concentration （IC50） was detcted by 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide. The expressions of Bcl-2 and caspase-3 were measured by real-time polymerase chain reaction and Western blot. By adenovirus transfection, A549/DTX-ad-PTEN cell line was constructed and inoculated subcutaneously into nude mice to develop implant-tumor. The tumor volume, growth rate and expressions of MDM2, Bcl-2 and caspase-3 were measured in A549/DTX xenografts and A549/DTX-ad-PTEN xenografts. Results The IC50 value of DTX in A549/DTX cells was significantly higher than that of parent strain A549 （ P 〈0.05）. PTEN gene expression was significantly lower than that of A549 cells and the expressions of Akt and MDM2 were significantly higher （all P 〈0.05）. The expression of PTEN was negatively correlated with the expression of MDM2. The overexpression of PTEN and MDM2 gene silencing reduced the IC50 value of DTX in A549/DTX cells, decreased the expression of Bcl-2 and increased the level of caspase-3 （all P 〈 0.05 ）.＇Also, the overexpression of PTEN induced the MDM2 gene silencing, reduced the tumor volume, slowed down the tumor growth rate, decreased the expression of Bcl-2 and increased the expression of caspase-3 （all P 〈 0.05 ）. Conclusions The overexpression of PTEN/Akt signaling pathway leads to high expression of MDM2, which can induce the over-proliferation of lung ad
作者
刘顺
单长波
郝敬林
刘磊
孟亚
Liu Shun;Shan Changbo;Hao Jinglin;Liu Lei;Meng Ya(Department of Respiratory Medicine, Lake West Hospital affiliated to Jining Medical College, Heze 274300, Chin)
出处
《国际呼吸杂志》
2018年第11期812-819,共8页
International Journal of Respiration
