香烟烟雾处理对BEAS-2B细胞FHIT基因甲基化及其mRNA表达的影响

Effects of methylation status and mRNA expression of FHIT on BEAS-2B cells exposed to cigarette smoke

目的观察永生化人支气管上皮细胞(BEAS-2B)经香烟烟雾长期处理后FHIT基因甲基化状况及其mRNA表达改变,探索吸烟致肺癌的机制。方法 BEAS-2B以20%烟雾浓度,每次10min作为染烟条件,细胞连续染烟2次作为染烟1代。细胞染烟30代且传代40代后,应用流式细胞仪分析细胞周期及凋亡和软琼脂克隆法检测克隆形成率判断其恶性转化程度,用甲基化特异性PCR(MS-PCR)检测FHIT基因甲基化状况,并通过Q-PCR检测FHIT的mRNA表达量。结果与对照组比较,各染烟组细胞出现了一定的S期阻滞,染烟后传代至40代细胞凋亡率较对照组下降明显(P<0.05)。染烟20代传代至40代细胞(Sm20-40)与染烟30代传代至40代细胞(Sm30-40)FHIT基因启动子区呈高甲基化状态,且Q-PCR结果显示Sm20-40和Sm30-40组细胞FHIT基因mRNA均低于对照组(P<0.05)。结论香烟烟雾可导致BEAS-2B细胞FHIT基因启动子区呈高甲基化状态,引起FHIT基因mRNA表达降低,可能是吸烟致肺癌的机制之一。

Objective To observe FHIT gene methylation and mRNA expression of immortalized human bronchial epithelial cells（ BEAS-2B） after long-term exposure to cigarette smoke,and to explore the mechanisms of smoking-induced lung cancer. Methods The BEAS-2B cells were exposed to smoke concentration of 20% for 10 min,and cells exposed to smoke twice as the exposure generation. After exposure for 30 passages and generation for 40 passages,using flow cytometry assay and soft agar cloning to determine the extent of its malignant transformation. FHIT gene methylation was detected by using Methylation-specific PCR（ MS-PCR）,and the FHIT mRNA expression was analyzed by using Q-PCR. Results Compared with the control cells,there was a certain S-phase arrest in each transfected cells exposed to smoking,the apoptosis rate of the cells of 40 passages after smoke exposure decreased significantly compared to control group（ P〈0. 05）. The FHIT gene promoter was hypermethylation of the cells after exposure for 20 passages and generation for 40 passages（ Sm20-40） and for 30 passages and generation for 40 passages（ Sm30-40）,the expression of FHIT gene in the Sm20-40 and Sm30-40 groups were lower than that in the control group（ P〈0. 05）. Conclusion Cigarette smoke can cause hypermethylation of FHIT of BEAS-2B cells,decrease the mRNA expression of FHIT,it may be one of the early mechanisms of smoking-induced lung cancer.