Temporal RNA-sequencing of cytologically-normal mouse airway epithelia during tobacco carcinogen-associated lung tumorigenesis

Smoking perpetuates in cytologically-normal airways a molecular “field of injury” that is pertinent to lung cancer and early detection. The evolution of airway field changes prior to lung cancer onset is poorly understood largely due to the long latency of lung malignancy in smokers. Here we studied airway expression changes prior to lung cancer onset in mice with knockout of the Gprc5a gene and tobacco carcinogen (nicotine-specific nitrosamine ketone; NNK) exposure and that develop the most common type of lung cancer, lung adenocarcinoma (LUAD). Cytologically-normal airway epithelial brushings were collected before exposure and at multiple times following NNK exposure until time of LUAD development and then analyzed by RNA-sequencing (RNA-Seq). Overall design: Groups of five to six eight week old mice (Gprc5a-/-) were intraperitoneally administered 50 mg/kg body weight NNK three times per week for 8 weeks. Normal airway (tracheal) epithelia were collected by brushing and studied at baseline (prior to NNK exposure) from six mice and immediately following the eight-week exposure (t=0) to understand early effects of tobacco carcinogen exposure on the airway transcriptome (Figure 1). Airway epithelia were also collected every two months following completion of NNK treatment (t=2, t=4, and t=6) and studied to understand progressive or lasting field effects following exposure and until time of LUAD development (t=6). In total, airway brushings (n=28) from 28 mice were sequenced.

吸烟会在细胞学正常的气道中维持一种与肺癌及早期检测相关的分子“损伤域”。目前人们对肺癌发生前气道域改变的演化过程尚不清楚，这在很大程度上源于吸烟者肺部恶性肿瘤存在较长的潜伏期。本研究针对敲除Gprc5a基因（Gprc5a gene）、暴露于烟草致癌物尼古丁特异性亚硝胺酮（nicotine-specific nitrosamine ketone; NNK）且可罹患最常见肺癌类型肺腺癌（lung adenocarcinoma, LUAD）的小鼠，探究其肺癌发生前的气道基因表达变化。研究人员在暴露前以及暴露于NNK后直至肺腺癌发生的多个时间点，收集细胞学正常的气道上皮刷检样本，随后通过RNA测序（RNA-sequencing, RNA-Seq）开展分析。
整体实验设计如下：将5~6只8周龄的Gprc5a基因敲除小鼠（Gprc5a knockout, Gprc5a-/-）按每周3次、共8周的方案，腹腔注射50 mg/kg体重剂量的NNK。选取6只小鼠在基线阶段（NNK暴露前）通过刷检获取正常气道（气管）上皮样本，并在8周暴露结束后即刻（t=0）取样，以解析烟草致癌物暴露对气道转录组的早期影响（图1）。此外，在NNK给药结束后，每隔2个月分别于t=2、t=4、t=6时间点收集气道上皮样本，以探究暴露后渐进性或持续性的气道域效应，并直至t=6时间点肺腺癌发生时终止取样。最终共获取28只小鼠的气道刷检样本（n=28）并完成测序。