Early Functional Genetic and Epigenetic Changes in Human Lung Carcinogenesis

描述（由申请人提供）：RFA-03-CA-101。 挑战领域：生物标志物发现和验证。 挑战主题：指纹用于癌症的早期检测和治疗。 题目：“人类肺癌发生的早期功能遗传学和表观遗传学变化。“肺癌的发生始于第一支香烟，但吸烟者导致早期疾病的基因失调事件的顺序和途径仍然不清楚。 我们的团队已经在表观基因组水平和基因座特异性水平上开发了几种检测方法，可以对基因失调进行独特的见解。 我们假设，在组织学上不同的肺上皮表型中进行转录组和表观基因组范围内的搜索将揭示候选的肺癌发生标记，其中一些将通过高分辨率检测被证实为肺癌发生的关键调控位点。 在肺癌病例受试者的初始和随后的确认组中，我们将生成初始发现转录组和可能具有表型意义的表观基因组范围标记，然后用高密度表观遗传参考技术对其进行验证和细化。 这将允许在DNA甲基化和microRNA水平上鉴定相关的表型定义表观基因组标记。 对于最初的基因表达变化，我们将通过表达微阵列检测（1）mRNA水平，然后通过实验室开发的RNA特异性mRNA-PCR进行验证。 为了测定表观基因组，我们将采用：（2）全甲基化HpaII微小片段富集连接介导的PCR（HELP）测定，通过使用基于质谱的测序进行更深入的测序来验证表型区分位点（MassArray（r）），并使用实验室开发的标记的亚硫酸氢盐基因组测序（tBGS）在关键基因座处重新验证;（3）通过平行测序进行的microRNA表达的全基因组测定，通过我们实验室开发的microRNA-qPCR和microRNA pull-down验证。 表达和表观遗传特征的关键一致标志物将在荧光支气管镜引导的刷片脱落的正常、异型增生和恶性上皮细胞中重新确认，这些细胞来自供体亚组。 因此，该项目将揭示在那些被证明具有这种倾向的个体（肺癌病例）中肺癌发生的不同步骤所固有的基因失调的全面和独特的模式。 这些研究是在未来由其他机制资助的前瞻性研究中开发风险和早期诊断气道生物标志物的前奏。 公共卫生相关性：该项目旨在发现基因表达和表观遗传基因调控特征，这些特征对于人类肺上皮从正常到癌前到癌的转变非常重要。 在平行研究中，我们正在积极开发非侵入性方法来测量人类中这些相同的表观遗传肺癌发生生物标志物，用于直接临床研究。目标是将表观遗传特征用于诊断、预防和治疗目的。

DESCRIPTION (provided by applicant): RFA-03-CA-101. Challenge Area: Biomarker Discovery and Validation. Challenge Topic: Fingerprints for the Early Detection and Treatment of Cancer. Title: "Early Functional Genetic and Epigenetic Changes in Human Lung Carcinogenesis." Lung carcinogenesis is initiated with the first cigarette, but the sequence and pathways of gene dysregulation events in smokers leading to early disease remain obscure. Our group has developed several assays at the epigenome-wide level and locus-specific level that allow unique insights into gene dysregulation. We hypothesize that a transcriptome and epigenome-wide search among histologically divergent lung-epithelial phenotypes will reveal candidate lung carcinogenesis marks, some of which will be confirmed by high resolution assays as key regulatory loci underlying lung carcinogenesis. In an initial and then confirmatory set of lung cancer case subjects, we will generate initial discovery transcriptome, and epigenome-wide marks of possible phenotypic significance that will then be verified and refined with high density epigenetic reference techniques. This will allow identification of relevant, phenotype defining epigenome marks at the DNA methylation, and microRNA levels. For initial gene expression changes, we will assay (1) mRNA levels by expression microarrays, that are then verified by laboratory-developed RNA specific mRNA-PCR. For assaying the epigenome, we will employ: (2) Methylome-wide HpaII tiny fragment Enrichment Ligation-mediated PCR (HELP) assay, with phenotype-differentiating loci verified by deeper sequencing using mass spectroscopy-based sequencing (MassArray(r)) and re-verified at key loci using the laboratory-developed tagged bisulfite genomic sequencing (tBGS); (3) Micronome-wide assays of microRNA expression by parallel sequencing, verified by both microRNA-qPCR and microRNA pull-down as developed in our laboratory. The key concordant marks for both expression and epigenetic features will be reconfirmed in fluorescent bronchoscopy-guided brush-exfoliated bronchial cells of normal, dysplastic, and malignant epithelium from a subset of donors. The project will therefore reveal comprehensive and unique patterns of gene dysregulation inherent to different steps of lung carcinogenesis in those individuals who prove to have that propensity (lung cancer cases). These studies are a prelude to the development of risk and early diagnostic airway biomarkers, in future prospective studies funded by other mechanisms. PUBLIC HEALTH RELEVANCE: This project is aimed at discovering gene expression and epigenetic gene regulation features that are important for the transition of human lung epithelium from normal to precancerous to cancerous. In parallel studies, we are actively developing non-invasive approaches to measuring these same epigenetic lung carcinogenesis biomarkers in humans for direct clinical studies. The goal is to use the epigenetic features for diagnostic, preventive, and therapeutic purposes.