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

描述(申请人提供)：除了烟草，酒精是头颈癌的主要危险因素。头颈部癌变的一些初始步骤被认为是表观遗传变化，它被定义为基因表达的变化，而不是潜在DNA编码序列变化的结果。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学改变，但是否由酒精在口腔上皮细胞中诱导全基因组表观遗传学改变尚不清楚。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。这项应用的目的是了解酒精是否调节全基因组的表观遗传学变化，如基因间隔区的DNA甲基化和组蛋白修饰，以及在人类口腔癌发生过程中表达变化的基因的启动子。我们还将确定维甲酸(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，在烟草中发现的酒精和致癌物会引起全基因组范围的异常表观遗传学变化，这将改变正常人类口腔上皮细胞的基因表达，而维甲酸和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学变化。两个特定的目标将检验这一假设：目标1：确定酒精是否调节表观遗传变化，以及这些变化是否与人类口腔上皮细胞基因表达的变化相关。乙醇处理的永生化口腔上皮细胞OKF6-TERT1的全基因组测序将在我们研究所表观遗传核心的帮助下进行。该核心在我们建议的方法方面拥有专业知识，其中包括：简化表示亚硫酸盐测序(RRBS)、染色质免疫沉淀测序(CHIP-SEQ)和RNA测序(RNA-SEQ)技术，分别确定DNA甲基化、组蛋白翻译后修饰和酒精处理后mRNA表达的全基因组变化。目的：探讨维甲酸和DNA甲基转移酶抑制剂对酒精和致癌物暴露后口腔上皮细胞表观基因组的调节作用。用B[a]P和乙醇处理的细胞将受到RRBS、CHIP-SEQ和RNA-SEQ的影响，以确定表观遗传学变化和基因表达的变化。我们还将研究苯并[a]磷和乙醇引起的表观遗传学变化是否可以通过RA和5-氮杂处理来逆转。这项研究具有创新性，因为它将使用最尖端的全基因组测序技术来研究表观遗传变化。这项拟议的研究具有重要意义，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，确定致癌物和酒精介导的口腔癌变过程中的一些初始步骤，从而为这种疾病提供更好的预防、预后和治疗方法。

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.