The Role of de novo Cytosine DNA Methyltransferases 3a & 3b in Gene Methylation

描述(由申请人提供)：肿瘤抑制基因的失活是所有形式的人类癌症发展的重要一步。在腺癌的早期癌前病变中，改变的基因和microRNAs(MiRs)及其调控的途径的阐明在很大程度上是未知的。恶性肿瘤和细胞系的特点是胞嘧啶DNA甲基转移酶(DNMT)1、DNMT3a和DNMT3b的表达增加。虽然已知DNMT在从头甲基化和基因沉默方面进行合作，但对DNMT3a和DNMT3b的靶点及其对腺癌发展的影响知之甚少。本课题组利用永生化的支气管上皮细胞系(HBECs)建立了肺癌癌前病变的体外转化模型。烟草致癌物可诱导HBECs发生形态转化，包括诱导上皮细胞向间充质细胞转化，表现为microRNA(MiR)-200家族和miR-205基因表达下调，胞嘧啶脱氧核糖核酸表达改变和肿瘤抑制基因甲基化。这一博士后研究解决了这样的假设，即DNMT3a和Dnmt3b(Dnevo Dnmts)针对特定的基因和miRs，在HBECs转化过程中进行甲基化和染色质沉默，并且DNMT3a和Dnmt3b的过表达将加速经致癌物质治疗的HBECs向完全恶性转化。将高表达DNMT3a或DNMT3b的HBECs暴露于烟草致癌物中，观察其对转化时间、转化效率、EMT及裸鼠体内生长的影响。[由于我们的初步数据表明Dnmt3b在HBEC2细胞中的过表达使转化效率提高了2倍，我们现在将重点放在Dnmt3b调控的基因上]。Illumina Infinium Human Methylation27珠芯片阵列将用于询问高表达HBECs与亲代HBECs或具有正常Dnmt3b背景的转化HBECs的Dnmt3b，以确定受Dnmt3b调控的甲基化基因。使用ILumina平台的下一代测序和用于芯片的定制平铺miR启动子阵列将用于评估与正常表达Dnmt3b的转化HBECs和双亲HBECs相比，在过度表达Dnmt3b的转化HBECs中存在的miR中染色质标记的分布。将在100个原发腺癌样本和17个肺癌衍生细胞系中检测8-10个Dnmt3b调节基因和2-3个miRs的启动子甲基化状态。两个在原发肿瘤中最常见的沉默基因和两个miR将在肺肿瘤来源的细胞系中过表达，并确定其对表型的影响。在这项奖学金的三年中，申请者将使用全基因组方法通过启动子甲基化阵列来询问表观基因组，通过下一代测序来询问微RNAome，并通过芯片分析来询问染色质重塑。为了加强他在生物信息学和统计学方面的教育，他将参加生物信息学的短期课程和为期一学期的生物统计学课程。这些活动以及通过本申请中概述的研究所接受的培训将为IVO提供独特的优势，以及整合分子生物学、生物信息学和统计学的能力。 公共卫生相关性：在美国，肺癌是一个巨大的公共卫生问题，导致了美国至少30%的癌症死亡，全球超过150万人死亡。参与这种疾病发展的基因仍在识别和表征中。本申请中描述的研究将识别参与肺癌发展的新基因和microRNAs，并将提供受这些基因影响的途径的基础知识。

DESCRIPTION (provided by applicant): Inactivation of tumor suppressor genes is an important step in the development of all forms of human cancer. The elucidation of altered genes and microRNAs (miRs) and the pathways they regulate that interact in early premalignancy of adenocarcinoma are largely unknown. Malignant tumors and cell lines are characterized by increased expression of cytosine DNA methyltransferase (DNMT) 1, DNMT3a and DNMT3b. While it is known that DNMTs cooperate in de novo methylation and gene silencing, very little is known about targets for DNMT3a and DNMT3b or their impact on the development of adenocarcinoma. Our group has developed an in vitro transformation model using immortalized bronchial epithelial cell lines (HBECs) to study pre-malignancy of lung cancer. Exposure to tobacco carcinogens induces morphological transformation involving induction of epithelial to mesenchymal transition in HBECs that manifests through down regulation of the microRNA (miR)- 200 family and miR-205, changes in expression of cytosine DNMTs and methylation of tumor suppressor genes. This Postdoctoral fellowship addresses the hypothesis that DNMT3a and DNMT3b (de novo DNMTs) target specific genes and miRs for methylation and chromatin silencing during transformation of HBECs, and that overexpression of DNMT3a and DNMT3b will accelerate transformation of carcinogen-treated HBECs to full malignancy. HBECs overexpressing DNMT3a or DNMT3b will be exposed to tobacco carcinogens, and the effect on time to transformation, transformation efficiency, EMT and growth in nude mice will be determined. [Since our preliminary data indicates that overexpression of DNMT3b in HBEC2 cells increased transformation efficiency by 2-fold, we will now focus on DNMT3b regulated genes]. The Illumina Infinium Human Methylation27 BeadChip array will be used to interrogate transformed DNMT3b overexpressing HBECs vs. parental HBECs or transformed HBECs with normal DNMT3b background to identify methylated genes regulated by DNMT3b. Next generation sequencing using the Ilumina platform and a custom tiling miR promoter array for ChIP-chip will be used to evaluate the distribution of chromatin marks in miRs present in transformed HBECs overexpressing DNMT3b compared to transformed HBECs with normal DNMT3b expression and parental HBECs. Promoter methylation status of 8-10 DNMT3b - regulated genes and 2-3 miRs will be determined in 100 primary adenocarcinoma samples and 17 lung tumor-derived cell lines. Two genes and two miRs that are most commonly silenced in primary tumors will be overexpressed in lung tumor-derived cell lines and the effect on phenotype determined. During the three years of this fellowship, the applicant will use whole genome approaches to interrogate the epigenome through promoter methylation arrays, the microRNAome through Next Generation sequencing, and chromatin remodeling through ChIP assays. To foster his education in bioinformatics and statistics, he will take a short course in bioinformatics and a one-semester course in Biostatistics. These activities along with the training received through the studies outlined in this application will provide Ivo with unique strengths, and the ability to integrate molecular biology, bioinformatics, and statistics. PUBLIC HEALTH RELEVANCE: Lung cancer is an enormous public health problem in the USA causing at least 30% of cancer deaths in the US, and over 1.5 million deaths globally. Genes involved in development of this disease are still being identified and characterized. The studies described in this application will identify new genes and microRNAs that participate in lung cancer development and will provide fundamental knowledge on the pathways affected by these genes.