| ObjectiveLung cancer is the leading cause of cancer-related deaths for both men and women worldwide. Tobacco intake is the main risk factor associated with lung cancer. Nicotinic acetylcholine receptor signaling pathway is the research focus of lung cancer in recent years.Genome-wide association analysis showed that CHRNA5 gene encoding a5-nicotinic acetylcholine receptor (a5-nAchR) is particularly relevant to lung cancer. However, the role of a5-nAchR and its mechanism in lung cancer are still the initial stage of the research. In this study, to investigate the optimal concentration and treatment time of nicotine on lung adenocarcinoma cell by using proliferation by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, such as A549, H1299 and H1975. Cell starvation model was constructed to study the downstream signaling molecule p-Src418, p-Raf340,341-1 p-Rb807 expression changes by Western blot. CHRNA5 siRNA fragment was constructed and then transfected into human lung cancer A549 cell line with lipofectamine 2000 to study the effect of CHRNA5 (cholinergic receptor, nicotinic, alpha5) gene silence on nicotine-induced lung cancer cell proliferation and the downstream signaling pathway.Methods1. Using direct sequencing to screen a5-nAchR SNP398 variants of lung adenocarcinoma cell lines and study the optimal concentration and treatment time of nicotine on lung adenocarcinoma cell by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.2. Cell starvation model was constructed to study the downstream signaling molecule p-Src418, p-Raf340,341-1, p-Rb807 expression changes by Western blot.3. CHRNA5 siRNA fragment was constructed and then transfected into human lung cancer A549 cell line with lipofectamine 2000. Expression of CHRNA5 mRNA and protein was examined by FQ-PCR and Western blotting analysis, respectively.4. To observed cell proliferation of A549 after CHRNA5 siRNA transfection using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.5. To comparatively analyze the changes of cell proliferation related molecules p-Src418, p-Raf340,341-1, p-Rb807 expression between nicotine treatment group and (RNAi interference+nicotine) group and explore the mechanism of a5-nAchR on cell proliferation.Results1. The SNP398 genotypes of A549, H1299 and H1975 were wild type (A/A), mutated-homozygote (A/G) and heterozygote (G/G) respectively. Nicotine accelerated the proliferation of lung adenocarcinoma cell lines in time-and dose-dependent manner. The optimal concentration and treatment time of nicotine on A549 and H1299 cell proliferation were luM/L for 24h (PA549<0.01, PH1299<0. 05) while was 24h treatment with 10uM/L nicotine on H1975 cell proliferation (P H1975<0.01)2. P-Src418 in 15 minutes, p-Raf340,341-1 in 2 hours, p-Rb807 at 6 hours were activated respectively in cells under a starvation state by 0.5% newborn calf serum for 48 hours compared with the control (P<0.05).3. CHRNA5 siRNA inhibited the expression of CHRNA5 mRNA and protein in A549 cells (P<0.05)4. Down-regulation of CHRNA5 showed a trend that the proliferation of A549 cells was inhibited. With nicotine treatment, CHRNA5 siRNA transfection significantly inhibited A549 cell proliferation compared with control group (P<0.01)5. Compared with the Nicotine treatment group, silence of CHRNA5 inhibited cell proliferation-related molecules p-Src418, p-Raf340,341-1, p-Rb807 (P<0.05) expression.Conclusions1. There are different sensitivities of lung adenocarcinoma with various a5-nAchR SNP 398 genetypes to nicotine.2. Down-regulation of CHRNA5 can inhibit nicotine-induced A549 cells proliferation which suggests that CHRNA5 may serve as a potential target for gene therapy of lung cancer.3. CHRNA5 involves in Raf/MAPK/ERK signaling pathway to promot non-small cell lung cancer cell proliferation which provides new experimental evidence for etiology of lung cancer. |
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