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目的:研究siRNA干扰尼古丁乙酰胆碱受体α5(cholinergic receptor nicotinic alpha5,CHRNA5)基因表达后对尼古丁促人肺癌A549细胞增殖作用的影响。方法:设计并合成CHRNA5-siRNA片段,并经脂质体介导转染A549细胞。荧光定量PCR、Western blotting检测转染后A549细胞中CHRNA5 mRNA和蛋白的表达,MTT法检测CHRNA5-siRNA对尼古丁促A549细胞增殖作用的影响。结果:成功构建了CHRNA5-siRNA并成功转染A549细胞。CHRNA5-siRNA转染组A549细胞的CHRNA5mRNA表达量明显低于si-NC转染对照组(0.196±0.044vs0.944±0.027,P<0.01),CHRNA5-siRNA转染组A549细胞的CHRNA5蛋白表达量明显低于si-NC转染组(0.267±0.031vs0.745±0.035,P<0.01)。在无尼古丁作用时,CHRNA5-siRNA的转染不能抑制A549细胞的增殖。在尼古丁作用下,A549细胞增殖显著增加(P<0.01),CHRNA5-siRNA转染可明显抑制尼古丁的促增殖作用(P<0.01)。结论:siRNA下调肺癌A549细胞中CHRNA5的表达可以抑制尼古丁促细胞增殖作用,CHRNA5可能是肺癌治疗的潜在靶点之一。
AIM: To investigate the effect of siRNA on nicotine-induced proliferation of human lung adenocarcinoma A549 cells induced by cholinergic receptor nicotinic alpha5 (CHRNA5) gene expression. Methods: The CHRNA5-siRNA fragment was designed and synthesized and transfected into A549 cells by liposome. The expression of CHRNA5 mRNA and protein in A549 cells was detected by real-time PCR and Western blotting. The effect of CHRNA5-siRNA on the proliferation of nicotine-stimulated A549 cells was detected by MTT assay. Results: CHRNA5-siRNA was successfully constructed and transfected into A549 cells successfully. The mRNA expression level of CHRNA5 in A549 cells transfected with CHRNA5-siRNA was significantly lower than that in si-NC transfected cells (0.196 ± 0.044vs0.944 ± 0.027, P <0.01) Significantly lower than the si-NC transfected group (0.267 ± 0.031vs0.745 ± 0.035, P <0.01). In the absence of nicotine, transfection of CHRNA5-siRNA did not inhibit the proliferation of A549 cells. The proliferation of A549 cells was significantly increased by nicotine (P <0.01). The transfection of CHRNA5-siRNA significantly inhibited the proliferation of nicotine (P <0.01). CONCLUSION: siRNA down-regulates the expression of CHRNA5 in lung cancer A549 cells and inhibits nicotine-induced cell proliferation. CHRNA5 may be one of the potential targets for the treatment of lung cancer.