胆管癌细胞Dicer募集HP1a调控RASSF1A启动子CpG位点从头甲基化的机制研究

The mechanism of de novo methylation of related genomic CpG loci in the process of malignant transformation of benign cells has been a bottleneck problem need to be resolved urgently for DNA methylation theory. Takes RASSF1A gene in cholangiocarcinoma cells as a study target, we discovered that the enrichment and interaction of Dicer and HP1a in promoter of RASSF1A gene impacts significantly on the heterochromatin state and methylation of CpG loci in cholangiocarcinoma cells. Based on both national and international research frontier and our pre-discoveries, this project puts forward the hypothesis that in the process of canceration of biliary epithelial cells, through recruiting HP1α, Dicer would regulate the spreading activity of heterochromatin and control the de novo methylation of RASSF1A promoter CpG loci. To deal with the key issues of this hypothesis, the emphasis is laid on 1. Dicer's regulation of both endogenous and exogenous RASSF1A promoter CpG loci's de novo methylation; 2. Dicer's influence on the spreading activity of heterochromatin; 3.The influence of heterochromatin spreading on methylation of CpG loci; 4.HP1α's role when Dicer is inducing heterochromatin spreading and de novo methylation of RASSF1A promoter CpG loci. This project mainly aims to illuminate the mechanism of de novo methylation of tumor-related CpG loci to enrich DNA methylation theory from the perspective of Dicer's regulation of heterochromatin spreading, and explore the strategy of molecular therapy for cholangiocarcinoma.

正常细胞恶性转化过程中，基因组相关CpG位点从头甲基化的发生机制，是甲基化理论亟待解决的"瓶颈"问题。为此，申请人以胆管癌细胞RASSF1A为研究对象，发现Dicer和HP1a蛋白在RASSF1A启动子的富集和偶联，对该区段染色质状态和CpG位点甲基化有显著影响。在此基础上并结合国内外研究前沿，项目提出科学假设：在胆管上皮细胞癌化过程中，Dicer募集HP1a，调节RASSF1A启动子区异染色质延伸，调控该区段CpG位点从头甲基化。针对科学假设的关键问题，项目重点研究：1. Dicer对内源性和外源性RASSF1A启动子CpG位点甲基化的调控；2.Dicer对异染色质延伸的调节；3.异染色质延伸对RASSF1A启动子CpG位点甲基化的影响；4.HP1a在Dicer调控RASSF1A启动子CpG位点从头甲基化中的作用。以探讨CpG位点从头甲基化机制，丰富甲基化理论，并探索胆管癌分子干预策略。

DNA甲基化在胆管癌等恶性肿瘤发生和进展中发挥重要作用。目前对于DNA甲基化发生后抑制基因表达的机制相对清楚，而在正常细胞恶性转化过程中，基因组相关CpG位点选择性地发生从头甲基化的机制尚不明了。本项目以Dicer为研究对象，实验发现，Dicer异常表达与特定肿瘤组织及临床特征相关。此外，研究显示在胆管癌中，Dicer主要定位于细胞核，并通过募集异染色质蛋白（HP1α），实现对相关基因的表观遗传学调控。该项目拟明胆管癌细胞Dicer介导HP1α调控SFRP1基因启动子从头甲基化的调控机制(SFRP1基因为实验过程中新增加的研究对象，与胆管癌表观遗传学关系密切，这是在研究计划允许的范围内进行的局部调整)。免疫组化发现，Dicer在胆管癌组织中主要定位于细胞核，且其高表达与肿瘤大小，淋巴结转移相关。免疫共沉淀（Co-IP）技术表明，Dicer特异性耦联HP1α，并募集H3K9me3/SUV39H1/Dnmts形成复合物。通过人类基因组450K甲基化芯片技术筛选出Dicer下调后呈低甲基化水平的候选基因SFRP1，经生物信息学分析，预测SFRP1基因启动子CpG岛包含28个CpG位点。亚硫酸氢盐测序发现，Dicer下调明显降低该启动子CpG岛的甲基化水平，并导致部分CpG位点发生去甲基化改变。染色质免疫共沉淀（ChIP）实验表明，Dicer/HP1α/H3K9me3选择性募集于SFRP1基因启动子区，抑制SFRP1的表达。此外研究表明，Dicer与SFRP1的表达呈明显的负相关，应用脱甲基化药物5-Aza-CdR和乙酰化酶抑制剂TSA抑制SFRP1的甲基化水平能重新恢复该基因的表达。同时生物学行为也显示，Dicer下调后能够抑制胆管癌细胞的增殖、侵袭及促进凋亡，联合下调SFRP1可部分恢复上述胆管癌细胞的生物学行为。我们的研究表明在肝门部胆管癌的发生和进展中，Dicer入核后特异性偶联HP1α，并募集组蛋白和DNA甲基化相关蛋白进而调控基因启动子CpG岛的从头甲基化，实现对相关基因的负性调控。