RNA-Seq studies identify cancer-related genes differentially regulated during inflammation-driven lung tumorigenesis and modulated by chemopreventive agents

Purpose: Chronic pulmonary inflammation in the form of chronic obstructive pulmonary disease (COPD) has been consistently shown to increase the risk of lung cancer. Therefore, identification of chemopreventive agents with anti-inflammatory effects, in addition to antiproliferative and apoptotic activities, is indispensable. Recently, we found that combinations of silibinin (Sil) and indole-3-carbinol (I3C) significantly inhibited lung tumorigenesis induced by 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK) and enhanced by chronic treatment with the inflammatory agent lipopolysaccharide (LPS). In this study, we described gene expression profiling of lung tissues using RNA-seq to determine the gene expression signature in inflammation-driven lung tumors and modulation of this signature by the chemopreventive agents Sil and I3C. Methods: Total RNA extracted from lung tissues of control and treated mice were processed for mRNA sequencing, in triplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed for transcript abundance at the gene level using CLC Bio Genomics Workbench and differential gene expression analysis was performed using the built-in Empirical Analysis of Differential Gene Expression (DGE) based on ''Exact Test'' method. qRT–PCR validation was performed using SYBR Green assays. Results and conclusions: We found that 330, 2,957, and 1,143 genes were differentially regulated in mice treated with NNK, LPS, and NNK + LPS, respectively. The expression of inflammation-and immunity-related genes was significantly more deregulated in lung tissues of mice treated with LPS alone compared to mice treated with NNK + LPS. Among 1,143 genes differentially regulated in the NNK + LPS group, the expression of 162 genes and associated signaling pathways were significantly modulated by I3C and/or Sil + I3C. These genes include cytokines, chemokines, and genes with a well-established role in inflammation and/or tumorigenesis such as c-ros oncogene 1 (Ros1), the EGFR ligands amphiregulin and epiregulin, Cyp1a1, and the circadian rhythm genes Arntl, and Npas2. To our knowledge, this is the first report that provides insight into genes that are differentially expressed during inflammation-driven lung tumorigenesis and the modulation of these genes by chemopreventive agents. Overall design: Lung tissue mRNA profiles of mice treated with control vehicle, NNK, LPS, NNK+LPS, or NNK+LPS supplemented with chemopreventive agent(s) were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000.

研究背景：以慢性阻塞性肺疾病（chronic obstructive pulmonary disease, COPD）为代表的慢性肺部炎症，已被多项研究证实会显著提升肺癌发病风险。因此，开发兼具抗炎、抗增殖及凋亡调控活性的化学预防剂至关重要。本团队前期研究发现，水飞蓟宾（silibinin, Sil）与吲哚-3-甲醇（indole-3-carbinol, I3C）联合使用，可显著抑制由4-(甲基亚硝氨基)-1-(3-吡啶基)-1-丁酮（4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone, NNK）诱导、且经慢性炎症刺激剂脂多糖（lipopolysaccharide, LPS）处理所增强的肺肿瘤发生过程。本研究通过RNA测序（RNA-seq）对小鼠肺组织进行基因表达谱分析，旨在明确炎症驱动型肺肿瘤的基因表达特征，以及化学预防剂Sil与I3C对该特征的调控作用。
实验方法：提取对照组与各处理组小鼠肺组织的总RNA，每组设置3次生物学重复，采用Illumina HiSeq 2000平台进行mRNA测序。对通过质量过滤的测序读段，使用CLC生物基因组工作站（CLC Bio Genomics Workbench）在基因水平分析转录本丰度；同时基于内置的基于“精确检验”（Exact Test）的差异基因表达实证分析（Empirical Analysis of Differential Gene Expression, DGE）工具完成差异基因表达分析。采用SYBR Green检测法进行定量实时聚合酶链反应（qRT–PCR）验证。
结果与结论：本研究发现，经NNK、LPS以及NNK+LPS分别处理的小鼠肺组织中，各有330、2957及1143个基因出现差异表达。与NNK+LPS处理组相比，单纯LPS处理组小鼠肺组织中炎症与免疫相关基因的表达失调程度更为显著。在NNK+LPS处理组的1143个差异表达基因中，有162个基因及其相关信号通路可被I3C或Sil+I3C显著调控。这些基因涵盖细胞因子、趋化因子，以及在炎症和/或肿瘤发生中已被广泛证实发挥作用的基因，例如c-ros癌基因1（c-ros oncogene 1, Ros1）、表皮生长因子受体（epidermal growth factor receptor, EGFR）配体双调蛋白（amphiregulin）与上皮调节蛋白（epiregulin）、细胞色素P450 1A1（Cyp1a1），以及昼夜节律相关基因Arntl与Npas2。据我们所知，本研究首次揭示了炎症驱动型肺肿瘤发生过程中差异表达的基因特征，以及化学预防剂对这些基因的调控作用。
实验整体设计：分别设置溶剂对照、NNK处理、LPS处理、NNK+LPS处理，以及NNK+LPS联合化学预防剂处理的小鼠组，每组设置3次生物学重复，采用Illumina HiSeq 2000平台进行深度测序，获取各组小鼠肺组织的mRNA表达谱。