mTOR通路抑制剂依维莫司对宫颈癌SiHa细胞恶性行为的影响

目的 探讨依维莫司（EVE）对人宫颈癌SiHa细胞增殖、凋亡及上皮间质转化（EMT）的影响。方法 常规培养SiHa细胞达对数生长期后，采用终浓度为1、10、100 μmol／L EVE处理SiHa细胞，采用四甲基偶氮唑盐比色法测定对照组及不同浓度EVE处理24、48、72和96 h后的细胞增殖情况，分别采用Annexin V-FITC／PI双染联合流式细胞术和实时荧光定量聚合酶链反应（qRT-PCR）检测不同浓度EVE处理96 h的早期和晚期凋亡率及凋亡相关基因（Bax、Bad和Bcl-2）的表达情况，Western blotting和免疫荧光法检测各组处理96 h后的EMT相关标记分子包括上皮型钙粘附素（E-cad）、纤维连接蛋白（FN）和波形蛋白（Vim）的水平。结果 EVE在1～100 μmol／L范围内对SiHa细胞有增殖抑制作用，且增殖抑制率呈剂量和时间依赖性，除1 μmol／L作用24 h外，其余浓度及作用时间的增殖抑制率均高于对照组（P＜0.05）；与对照组相比，EVE处理96 h后的早期、晚期凋亡率及Bax、Bad的mRNA水平均升高，而Bcl-2 mRNA水平降低，差异均有统计学意义（P＜0.05），且呈剂量依赖性；EVE处理后的E-cad水平均高于对照组，而FN和Vim水平均低于对照组（P0.05）。结论 采用EVE阻断mTOR通路对宫颈癌SiHa细胞有毒性作用，不仅抑制其增殖且诱导凋亡，可能与抑制其EMT过程有关。

Effect of the mTOR inhibitor everolimus on the malignant behavior of cervical cancer SiHa cells

Objective To investigate the effect of an mTOR pathway inhibitor everolimus（EVE） on the proliferation， apoptosis and epithelial mesenchymal transition（EMT） of human cervical cancer SiHa cells. Methods After being cultured in normal culture， the SiHa cells were treated with EVE at final concentrations of 1， 10 and 100 μmol／L. The cell proliferation was measured by methyl thiazolyl tetrazolium in the control group and different concentrations of EVE at 24， 48, 72 and 96 h after exposure. The early and late apoptosis rate was measured by Annexin V／PI double staining via flow cytometry， and the apoptosis related gene expression（Bax， Bad and Bcl-2） was assessed by real time fluorescence quantitative polymerase chain reaction. Western blotting and immunofluorescence assay were used to detect the levels of EMT related marker molecules， including E cadherin（E-cad）， Fibronectin（FN） and Vimentin（Vim） at 96 h after EVE exposure. ResultsEVE had inhibitory effect on the proliferation of SiHa cells in the range of 1 100 μmol／L in a dose and time dependent manner. Except for 1 μmol／L at 24 h， the proliferation inhibition rates of the remaining concentration and action times were higher than that of the control group（P＜0.05）. Compared with the control group， EVE treatment for 96 h could dose dependently increase the apoptosis rate and mRNA levels of Bax and Bad but decrease Bcl 2 level（P＜0.05）. Compared with the control group， the level of E cad was increased but the levels of Vim and FN were decreased after treatment with different concentrations EVE for 96 h（P＜0.05）. Conclusion Blocking mTOR pathway by EVE has a toxic effect on cervical cancer SiHa cells， which not only inhibit the proliferation and induce apoptosis， but may be related to the inhibition of EMT process.