CHRNA5基因表达下调对肺癌细胞VEGF表达的影响

Effect of down-regulated CHRNA5 gene expression on VEGF expression of lung cancer

目的体外研究非小细胞肺腺癌A549细胞中CHRNA5基因表达下调对尼古丁促肺癌细胞增殖及VEGF表达的影响。方法实验分为0μmol/L尼古丁组、1μmol/L尼古丁组、转染阴性对照组(1μmol/L尼古丁+si-NC)和转染组(1μmol/L尼古丁+CHRNA5-siRNA)。设计合成编码α5-nAChR的CHRNA5基因siRNA片段,脂质体介导转染A549细胞,RT-PCR检测CHRNA5-siRNA转染后α5-nAChR和VEGF mRNA水平的表达,Western blotting检测转染后α5-nAChR蛋白水平的表达,ELISA法检测转染后细胞上清液中VEGF蛋白水平的表达,CCK-8方法检测α5-nAChR下调对尼古丁促肺癌细胞增殖的影响。结果 CHRNA5-siRNA转染A549细胞后,转染组α5-nAChR和VEGF mRNA表达和蛋白表达量明显低于转染阴性对照组(P<0.05),且显著抑制尼古丁促肺癌细胞的增殖(P<0.05)。结论α5-nAChR表达下调能抑制尼古丁促肺癌细胞的增殖,降低VEGF的表达。α5-nAchR及其信号转导系统在吸烟相关性肺癌发生、发展中可能发挥重要作用,可作为肺癌预后预测指标和治疗靶点。

Objective To explore the influence of small interfering RNA （siRNA） down-regulating α5-nAChR expres- sion on nicotine-induced proliferation of human lung cancer cells and expression of VEGF in vitro. Methods The cells were divided into 0 μmol/L nicotine group, 1 μmol/L nicotine group, si-NC group （ 1 μmol/L nicotine ＋ si-NC ） and siRNA group （ 1 μmol/L nicotine ＋ CHRNAS-siRNA）. siRNA fragment of CHRNA5 encoding α5-nAChR was con- structed and transfected into human lung cancer A549 cell line with lipofectamine 2000. After CHRNAS-siRNA trans- fection, expressions of α5-nAChR and VEGF in A549 cell were detected by RT-PCR, Western blotting and ELISA. The nicotine-induced A549 cell proliferation was detected by CCK-8. Results After CHRNAS-siRNA transfection, expressions of α5-nAChR and VEGF on mRNA and protein levels were significantly decreased compared with the si-NC group （ P 〈 0.05 ）, and nicotine-induced A549 cell proliferation was significantly inhibited （ P 〈 0.05 ）. Conclusion Down-regulation of ctS-nAChR expression could inhibit nicotine-induced cell proliferation and decrease VEGF expres- sion of lung cancer, ctS-nAChR and its signal transduction system may play an important role in the development of smoking-related lung cancer and can be used as predictors of lung cancer prognosis and therapeutic targets.