雷帕霉素对肺癌A549细胞球增殖和上皮-间质转化的作用研究

Effects of rapamycin on proliferation and epithelial-mesenchymal transition of lung cancer A549 cells-derived spheres

目的：研究雷帕霉素对肺癌干细胞球的作用及其相关机制。方法：应用肿瘤干细胞培养液培养肺癌A549细胞球,并采用FCM法检测细胞球中CD133＋CD44＋细胞的比例。应用20、50和100 ng/m L的雷帕霉素分别干预A549细胞球,蛋白质印迹法检测雷帕霉素对磷酸化哺乳类动物雷帕霉素靶蛋白（phospho-mammalian target of rapamycin,p-m TOR）表达的抑制程度,选出最佳抑制浓度。应用最佳浓度的雷帕霉素干预A549细胞球24 h后,采用蛋白质印迹法检测E-钙黏蛋白（E-cadherin）、波形蛋白（vimentin）、磷酸化信号转导和转录激活因子3（phospho-signal transducer and activator of transcription 3,p-STAT3）、磷酸化p70核糖体蛋白S6激酶（phospho-p70 ribosomal protein S6 kinase,p-p70S6K）和磷酸化真核细胞始动因子4E结合蛋白1（phospho-4E binding protein 1,p-4EBP1）的表达水平,实时荧光定量PCR法检测Sox2、Oct4、CD133和Nanog的m RNA水平。MTT法检测雷帕霉素干预1～7 d后细胞的增殖能力,并应用相差显微镜观察第14天时细胞球的大小。结果：成功培养出悬浮生长的A549细胞球,FCM法检测该细胞球中CD133＋CD44＋细胞所占百分率为（75.0±8.7）%。20、50和100 ng/m L的雷帕霉素均可抑制p-m TOR蛋白的表达（P〈0.05）,其中100 ng/m L的抑制效果最好。100 ng/m L雷帕霉素干预A549细胞球24 h后,E-cadherin蛋白的表达水平明显升高（P〈0.05）,而vimentin、p-STAT3、p-p70S6K和p-4EBP1蛋白的表达水平以及Sox2、Oct4、CD133和Nanog的m RNA水平均明显降低（均P〈0.05）。雷帕霉素干预A549细胞球,在第3～7天时显著抑制细胞生长,第14天时细胞球体明显减小。结论：雷帕霉素可抑制A549细胞球的细胞增殖、成球能力及上皮-间皮转化,它可能是通过m TOR-STAT3和p70S6K等多种信号通路来发挥作用的。

Objective： To study the effect of rapamycin on lung cancer stem cell-derived spheres, and explore its related mechanism. Methods： The lung cancer A549 cell-derived spheres were obtained by culture with tumor stem cell conditioned medium. The proportion of CD133^＋ CD44^＋ cells in A549 cell- derived spheres was detected by flow cytometry. Then the spheres were treated with three concentrations （20, 50 and 100 ng/mL） of rapamycin, respectively. After that, the inhibition effect of rapamycin on the expression of phospho-mammalian target of rapamycin （p-mTOR） was detected by Western blotting, and the optimal inhibition concentration of rapamycin was selected for further research. After A549 cell-derived spheres were treated with 100 ng/mL rapamycin for 24 h, the expression levels of E-cadherin, vimentin, phospho-signal transducer and activator of transcription 3 （p-STAT3）, phospho-p70 ribosomal protein $6 kinase （p-p70S6K） and phospho-4E-binding protein 1 （p-4EBP1） were detected by Western blotting, while the mRNA levels of SRY （sex-determining region Y）-box 2 （Sox2）, Oct4, CD133 and Nanog were examined by real-time fluorescent quantitative-PCR. The proliferative activities of A549 cells after rapamycin treatment for 1-7 days were detected by MTT method, and the size of the spheres after rapamycin treatment for 14 d was observed by contrast microscopy. Results： The A549 cell-derived spheres were successfully obtained by suspension culture, and proportion of CD133^＋ CD44^＋ cells in the spheres was （75.0＋8.7）%. All three concentrations of rapamycin （20, 50 and 100 ng/mL） inhibited the expression of p-mTOR protein, and 100 ng/mL was the optimal inhibition concentration （P 〈 0.05）. After treatment with 100 ng/mL rapamycin for 24 h, the expression level of E-cadherin protein in A549 cell-derived spheres was obviously increased （P 〈 0.05）, but the expression levels of vimentin, p-STAT3, p-p70S6K and p-4EBP1 proteins were decreased （P 〈 0.05）, and the levels of Sox2, Oct4, CD133 and Nanog mRNAs were also decreased （P 〈 0.05）. Rapamycin could significantly inhibit the proliferation of A549 cells during 3-7 days, and the size of the spheres on the 14th day was smaller than that of the spheres without rapamycin treatment. Conclusion： Rapamycin can inhibit the proliferation of A549 cell-derived spheres, the sphere-formation ability and epithelial-mesenchymal transition. The mTOR-STAT3 and p-70S6K signaling pathways may play important roles in these processes.