Cigarette Smoking Induces Small Airway Epithelial Epigenetic Changes with Corresponding Modulation of Gene Expression

The small airway epithelium (SAE), the first site of smoking-induced lung pathology, exhibits genome-wide changes in gene expression in response to cigarette smoking. Based on the increasing evidence that the epigenome can respond to external stimuli in a rapid manner, we assessed the SAE of smokers for genome-wide DNA methylation changes compared to nonsmokers, and whether changes in SAE DNA methylation were linked to the transcriptional output of these cells. Using genome-wide methylation analysis of SAE DNA of nonsmokers and smokers, the data identified 204 unique genes differentially methylated in SAE DNA of smokers compared to nonsmokers, with 67% of the regions with differential methylation occurring within 2 kb of the transcriptional start site. Among the genes with differential methylation were those related to metabolism, transcription, signal transduction and transport. For the differentially methylated genes, 34 exhibited a correlation with gene expression, 53% with an inverse correlation of DNA methylation with gene expression and 47% a direct correlation. These observations provide evidence that cigarette smoking alters the DNA methylation patterning of the SAE and that, for some genes, these changes are associated with the smoking-related changes in gene expression. Small airway epithelium DNA was assessed for genome-wide methylation using the microarray-based high resolution HpaII tiny fragment enriched by ligation-mediated PCR (HELP) assay. Small airway epithelium transcriptome was also assessed for healthy nonsmokers (n=16) and healthy smokers (n=20) with Affymetirx HG-U133 Plus 2.0 arrays

小气道上皮（small airway epithelium, SAE）作为吸烟诱导肺部病变的首发部位，其基因表达会因吸烟暴露而出现全基因组范围的改变。鉴于越来越多研究证据表明表观基因组可快速响应外界刺激，本研究对比了吸烟者与非吸烟者的小气道上皮，检测其全基因组DNA甲基化变化，并探究小气道上皮的DNA甲基化改变是否与这些细胞的转录产物水平相关。通过对非吸烟者与吸烟者的小气道上皮DNA开展全基因组甲基化分析，本研究共鉴定出204个在吸烟者小气道上皮DNA中存在差异甲基化的独特基因，其中67%的差异甲基化区域位于转录起始位点（transcriptional start site）上下游2kb范围内。差异甲基化基因涉及代谢、转录、信号转导及物质转运等生物学过程。在上述差异甲基化基因中，有34个与基因表达存在相关性：其中53%表现为DNA甲基化水平与基因表达量呈负相关，47%呈正相关。上述研究结果证实，吸烟可改变小气道上皮的DNA甲基化模式，且部分基因的此类甲基化改变与吸烟诱导的基因表达变化存在关联。本研究采用基于微阵列的高分辨率连接介导PCR富集HpaII微小片段（HELP）检测法，对小气道上皮DNA开展全基因组甲基化分析。同时，本研究使用Affymetrix HG-U133 Plus 2.0基因芯片，对16名健康非吸烟者与20名健康吸烟者的小气道上皮转录组（transcriptome）进行了检测。