dsRNA激活PTEN基因对肺癌细胞生物活性的影响

Cell biologic changes in thd cells which PTEN gene activated by double-stranded

目的探讨双链RNA分子（dsRNA）促进肺癌细胞中PTEN基因表达后，肺癌细胞生物活性的改变。方法根据前期研究结果，利用针对PTEN基因启动子区域非CpG岛序列的dsRNA，将其转染入肺癌肿瘤细胞A549和H292，绘制生长曲线，探讨转染后细胞增殖变化通过Transwell小室法检测侵袭能力，以及通过流式细胞仪检测细胞周期的变化。结果A549和H292细胞转染特异dsRNA分子后，与未经转染的细胞相比，PTEN基因mRNA表达量可增至4倍，但细胞的增殖、侵袭能力无显著变化。与对照组比，较多细胞停留于G1期。结论dsRNA分子激活后可以增加PTEN基因表达，但对于其细胞功能并无显著影响。

Objective To evaluate thd cell biologic changes in thd non-small-cell lung cancer（NSCLC） which PTEN gene were activated by double-stranded RNA （dsRNA）. Methods Specific dsRNA was designed. First, the promoter region of PTEN gene was determined by Promoter 2.0 program, then the CpG island in the promoter was found by CpGisland searcher software and the possible target non-CpG sequence that dsRNA might activate were defined by SiRNA Target Finder software. dsRNA were synthesized at Genechem Company（ Shanghai, China）. Then the specific dsRNA was transfeeted into A549 and H292 ceils which were stored in our laboratory using Lipofeetamine 2000 （ Invitrogen, USA） according to manufaeture＇s instruction. Total celluar RNA was isolated. The expression of PTEN mRNA in transfected, control and mock group were determined by real-time quantitative polymerase chain reaction. Cell profiferation was investigated on days 1 to 5 by using Cell Counting Kit-8 according to the manufature＇s technical manual. Cell invasion ability was assessed by Transwell method that transmembrane cells were counted, and cell bycle distribution were studied by flow cytometer （FCM） using CycleTESTTM PLUS DNA Reagent Kit. Results After the introduction of dsRNA into the A549 cells, the PTEN mRNA expressin was upregnlated to （ 4.35 ± 0.42 ） folds compared with the mock and control cells. And in H292 cells, the mRNA expression of PTEN was upregnlated to （3.92 ± 0.20） folds. It confirmed the RNA activation phenomenon in the PTEN gene in NSCLC cells. Compared with the control group, the number of alive transfeeted cells did not decreased in the cell proliferation assay. In the cell invasion test we found that the transmembrane A549 cells were 122.4± 11.2 vs. 150.7 ± 13.1 in transfected group and control group respectively. In the cell cycle distribution we found dsRNA in duced part of the transfected cells arrested in G1 phase and a corresponding decrease in S-phase population was observed, though this change was not statistically significant. Conclusion The expression of PTEN mRNA could by enhanced by inducing the specific dsRNA into the A549 and H292 cells, though no evidence was found that after the activation of silenced PTEN, the cell proliferation and invasion ability were significantly changed.