基于高通量测序的前列腺癌耐药相关候选DNA甲基化基因鉴定与功能研究

Prostate cancer is the most common urinary tract tumors in men. The emergence of progressive androgen-independent phenotypes is an important sign that prostate cancer is undergoing further deterioration after androgen ablation, and the underlying mechanism involved is still unclear. DNA methylation is an important epigenetic modification involved in the control of gene expression and prostate cancer. In our previous work, genome wide DNA methylation and transcriptome was analyzed in androgen-dependent prostate cancer cells (LNCaP) and androgen-independent prostate cancer cells (LNCaP-AI) using the Solexa technology, it was found that many genes were associated with drug-resistant prostate cancer (EPHA3, IGF-IR, TM4SF1, CASP8, DAPK1). We will validate candidate genes DNA methylation status by bisulphite sequencing with the goal of identifying key genes involved in prostate cancer progression. These genes will be down-regulated and up-regulated by transfection technology in prostate cancer cells. To understand the function of gene is to observe biological changes, such as the sensitivity to androgen and Flutamide, cell proliferation and apoptosis. Our study is aimed to identify some key genes involved in prostate cancer androgen-independent conversion process which will provide a foundation for future study.

前列腺癌是男性泌尿系最常见肿瘤之一，经内分泌治疗后易转化为雄激素非依赖性前列腺癌，给临床治疗带来困难且其转化机制尚不清楚。DNA甲基化作为一种重要的表观遗传修饰系统，在基因表达调控中扮演了关键的角色，与前列腺癌的发生密切相关。本课题前期采用Solexa高通量测序技术对雄激素依赖的前列腺癌细胞(LNCaP)和雄激素非依赖的前列腺癌细胞(LNCaP-AI)进行全基因组甲基化和转录组测序，发现多个可能与前列腺癌耐药相关的DNA甲基化基因(EPHA3；IGF-IR；TM4SF1；CASP8；DAPK1)。我们将采用重亚硫酸盐测序验证候选基因DNA甲基化水平，确定与前列腺癌耐药相关的甲基化基因。通过细胞转染方法上调或干扰候选基因表达来检测其对前列腺癌细胞雄激素敏感性、氟他胺耐受性、细胞增殖与凋亡等指标的变化，获得调控雄激素非依赖转变过程的功能基因，为更深入探索雄激素非依赖性前列腺癌发生机制奠定基础。

本研究旨在从细胞水平探究前列腺癌雄激素非依赖表型的形成机制。应用DNA甲基化免疫共沉淀测序技术分别检测雄激素非依赖性前列腺癌细胞（LNCaP-AI-F）及雄激素依赖性前列腺癌细胞（LNCaP）的甲基化情况。筛选差异甲基化区域（Differentially methylated regions, DMRs），并预测可能与DMRs相关的转录因子。通过整合甲基化数据及我们前期获得的RNA及miRNA转录组数据，筛选异常甲基化的差异表达基因（Differentially expressed genes with DMRs, MDEGs）及miRNA（Differentially expressed miRNAs with DMRs, MDEmiRNAs）。我们将那些启动子区与DMRs有重合的差异表达基因（Differentially expressed genes, DEGs）定义为PMDEGs。预测PMDEGs的潜在转录因子，并构建转录因子-靶基因调控网络。预测MDEmiRNAs所调控的基因，构建miRNA-靶基因调控网络。随后，对去雄激素培养基中驯化的基因表达谱芯片进行时间序列分析。此外，对DMRs、MDEGs和DEmiRNAs进行了验证。一共发现了18447个DMRs、3369个MDEGs、850个PMDEGs及1个MDEmiRNA （即miR-429）。构建了一个转录因子-靶基因调控网络，包括94个PMDEGs和5个转录因子。构建了一个miRNA-靶基因调控网络，包括172个MDEGs和miR-429。基于时间序列分析网络中的基因，发现NEDD4L和PBX3被SOX5靶向调控，而GNAQ、ANLN、KIF11被miR-429靶向调控。通过实时定量PCR，验证了这些基因和miR-429的表达水平。此外，通过公共数据集验证了109个DMRs。调控互作通路SOX5-NEDD4L/PBX3、miR429-GNAQ/ANLN--RHOA及miR429-ANLN--KIF11可能参与前列腺癌细胞雄激素非依赖表型的形成。