Molecular Analysis of Alcohol Effects on Human Oral Epithelium Cells' Epigenome

描述（由申请人提供）：除了烟草，酒精是头颈癌的主要危险因素。头颈癌发生的一些初始步骤被认为是表观遗传变化，表观遗传变化被定义为基因表达的变化，而不是潜在DNA编码序列改变的结果。酒精及其代谢物乙醛（AcH）促进乳腺癌、肝癌和结肠癌的表观遗传变化，但酒精是否诱导口腔上皮细胞的全基因组表观遗传变化尚不清楚。这些研究的长期目标是了解酒精促进头颈部肿瘤发生的分子机制。本申请的目的是了解酒精是否调节全基因组表观遗传变化，如基因间区域和人类口腔癌发生过程中表达发生变化的基因启动子的DNA甲基化和组蛋白修饰。我们还将确定视黄酸（RA）和DNA甲基转移酶抑制剂5-氮杂-2 '-脱氧胞苷（5-Aza）是否可以逆转烟草致癌物苯并[a]芘（B[a]P）和酒精诱导的表观遗传变化。我们的中心假设是，酒精和烟草中发现的致癌物将诱导全基因组异常表观遗传变化，这将改变正常人类口腔上皮细胞的基因表达，视黄酸和DNA甲基转移酶抑制剂将逆转酒精和致癌物相关的表观遗传变化。两个具体的目标将测试这一假设：目的1：以确定是否酒精调节表观遗传的变化，如果这些变化与人类口腔上皮细胞的基因表达的变化。乙醇处理的永生化OKF 6-TERT 1人口腔上皮细胞的全基因组测序将在我们研究所的表观遗传核心的协助下进行。该核心在我们提出的方法中具有专业知识，其中包括：还原亚硫酸氢盐测序（RRBS），染色质免疫沉淀测序（ChIP-seq）和RNA测序（RNA-seq）技术，分别用于确定DNA甲基化，组蛋白翻译后修饰和酒精处理后mRNA表达变化的全基因组变化。目标二：确定RA和DNA甲基转移酶抑制剂在酒精和致癌物暴露后调节人口腔上皮细胞表观基因组中的作用。用B[a]P和乙醇处理的细胞将经受RRBS、ChIP-seq和RNA-seq以确定表观遗传变化和基因表达的变化。我们还将研究是否B[a]P和乙醇诱导的表观遗传变化可以逆转RA和5-Aza治疗。这项研究是创新的，因为它将使用最先进的全基因组测序技术来研究表观遗传变化。拟议的研究是重要的，因为它将完成国家牙科和颅面研究所（NIDCR）和国家酒精滥用和酒精中毒研究所（NIAAA）的使命，通过确定致癌物和酒精介导的口腔癌发生过程中的一些初始步骤，这将导致更好的预防，预后和治疗方法，这种疾病。 公共卫生相关性：这项拟议中的研究与公共卫生有关，因为它将为酒精如何促进头颈癌提供一种机制。这些研究还将使我们能够确定在癌症发生和发展的最初步骤中细胞中发生的变化，并确定某些药物治疗是否可以逆转酒精引起的变化。因此，这些研究将有助于更好地预防、诊断和治疗头颈癌。

DESCRIPTION (provided by applicant): In addition to tobacco, alcohol is the major risk factor for head and neck cancer. Some of the initial steps of head and neck carcinogenesis are believed to be epigenetic changes, which are defined as changes in gene expression that are not a result of alterations in the underlying DNA coding sequence. Alcohol and its metabolite acetaldehyde (AcH) promote epigenetic changes in breast, liver, and colon cancers, but whether or not genome-wide epigenetic changes are induced by alcohol in oral epithelial cells is unknown. The long term goal of these studies is to understand the molecular mechanisms by which alcohol promotes head and neck tumorigenesis. The objective of this application is to understand whether alcohol regulates genome-wide epigenetic changes, such as DNA methylation and histone modifications in intergenic regions and at the promoters of genes whose expression is changed during human oral carcinogenesis. We will also determine whether retinoic acid (RA) and the DNA methyltransferase inhibitor 5-Aza-2'-deoxycitidine (5-Aza) can reverse epigenetic changes induced by the tobacco carcinogen benzo[a]pyrene (B[a]P) and alcohol. Our central hypothesis is that alcohol and carcinogens found in tobacco will induce genome-wide aberrant epigenetic changes that will alter gene expression of normal human oral epithelial cells, and that retinoic acid and DNA methyltransferase inhibitors will reverse alcohol and carcinogen associated epigenetic changes. Two specific aims will test this hypothesis: Aim 1: To determine if alcohol regulates epigenetic changes and if these changes correlate with changes in gene expression in human oral epithelial cells. Genome-wide sequencing of ethanol treated immortalized OKF6-TERT1 human oral epithelial cells will be performed with the assistance of our institute's Epigenetic Core. This core has expertise in our proposed approaches which include: Reduced Representation Bisulfite Sequencing (RRBS), Chromatin Immunoprecipitation sequencing (ChIP-seq), and RNA-sequencing (RNA-seq) techniques, to determine genome-wide changes in DNA methylation, histone post-translational modifications, and changes in mRNA expression after alcohol treatment, respectively. Aim 2: To determine the role of RA and DNA methyltransferase inhibitors in regulating the epigenome of human oral epithelial cells after alcohol and carcinogen exposure. Cells treated with B[a]P and ethanol will be subjected to RRBS, ChIP-seq, and RNA-seq to determine epigenetic changes and changes in gene expression. We will also examine whether B[a]P and ethanol induced epigenetic changes can be reversed by RA and 5-Aza treatment. This research is innovative because it will use the most cutting edge genome-wide sequencing techniques used to study epigenetic changes. The proposed research is significant as it will fulfill the mission of the National Institute of Dental and Craniofacial Research (NIDCR) and the National Institute of Alcohol Abuse and Alcoholism (NIAAA) by identifying some of the initial steps during carcinogen and alcohol mediated oral carcinogenesis, which will lead to better preventative, prognostic, and treatment approaches for this disease. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because it will provide a mechanism for how alcohol can promote head and neck cancers. These studies will also allow us to identify changes that occur in cells during the initial steps of cancer initiation and progression, and to determine whether certain drug treatments can reverse the changes induced by alcohol. As a result, these studies will allow for better prevention, diagnosis, and treatment of head and neck cancers.