gga-miR-130b-3p及其启动子DNA甲基化影响鸡马立克氏病肿瘤转化的分子机制

从表观基因组学角度研究宿主对于马立克氏病（Marek’s Disease, MD）的抗性/易感机制可为家禽抗病育种提供理论基础。前期申请者已发现在MD肿瘤化脾脏中DNA甲基转移酶基因表达量升高，gga-miR-130b-3p编码基因的启动子区域高甲基化是引发该miRNA在MD肿瘤化脾脏中显著低表达的原因，并且该miRNA能抑制MD肿瘤细胞增殖、迁移和侵袭。本研究将深入解析gga-miR-130b-3p编码基因的启动子区域差异甲基化的作用机制，通过双荧光素酶报告基因系统、去甲基化处理、DNA pulldown联合质谱分析和凝胶迁移等功能基因组学技术在细胞水平上确定调控该miRNA表达的CpG岛位点和结合的转录因子，同时利用基因功能缺失验证该miRNA的靶基因，并检测该miRNA和相应的靶基因对MD肿瘤细胞特性的影响，以揭示gga-miR-130b-3p在MD肿瘤转化过程中的作用机制。

Studying the host’s resistance/susceptibility to Marek’s Disease (MD) from the perspective of epigenomics can provide a theoretical basis for disease-resistant breeding in poultry. In previous experiments, we have found that the expression levels of DNA methyltransferase genes were increased in MD tumorous spleens. The promoter region of gga-miR-130b-3p encoding gene is hypermethylated, which induced its downregulation in MD tumorous spleens. In addition, this miRNA could inhibit the proliferation, migration, and invasion of MD tumorous cells. In this study, we will further study the mechanism of gga-miR-130b-3p encoding gene with differential methylation levels in the promoter region. We will determine the CpG island sites in the promoter that regulates the expression of this miRNA and bounded transcription factor at cell level using dual-luciferase reporter gene system, demethylation of MD tumorous cells, DNA pulldown-MS analysis, and EMSA. Furthermore, we will verify the target gene of gga-miR-130b-3p by loss of gene’s function and analyze the effects of gga-miR-130b-3p and its target gene on MD tumorous cellular features, to reveal the mechanism of this miRNA in MD tumor transformation process.