Dicer-HP1α-MSEs通路调控胆管癌细胞RASSF1A启动子从头甲基化的机制

Exploring the mechanism of de novo methylation of tumor-related genomic CpG loci in the malignant transformation process of nomal cells has been a challenge for DNA methylation theory. We have shown that Dicer significantly affects the de novo methylation of CpG loci in RASSF1A promoter through recruiting HP1α to form a Dicer-HP1α complex. However, the Dicer-HP1α complex does not directly recruit endogenous de novo methyltransferase. So, identifying the related methylation silencing effectors (MSEs) to map Dicer-HP1α-MSEs pathway has been a key issue need to be resolved urgently. For this purpose, we have utilized human genome-wide shRNA library and found 20 candidate MSEs. Based on our pre-discoveries, the current project will focus on: 1) identifying the MSEs downstream of HP1α; 2) elucidating the sequential recruitment of MSEs to RASSF1A promoter and thus mapping the Dicer-HP1α-MSEs pathway; 3) validating the Dicer-HP1α-MSEs pathway in HP1α knockout and transgenic animal model. We aim to explore the mechanism of de novo methylation of tumor-related CpG loci, as well as the strategy of molecular therapy in 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，是甲基化理论的瓶颈问题。申请人前期在胆管癌细胞中发现：Dicer通过募集HP1α形成Dicer-HP1α复合物，对RASSF1A启动子CpG位点从头甲基化具有显著调控作用。但是，Dicer-HP1α复合物并不能直接募集从头甲基转移酶，因此确定相关甲基化沉默因子（MSEs）并建立完整的调控通路，成为亟待澄清的关键问题。为此，我们利用人基因组shRNA文库筛选出20种候选MSEs。本项目在前述基础上，拟重点研究：1.鉴定HP1α下游MSEs种类；2.确定MSEs被募集的先后顺序，建立Dicer-HP1α-MSEs调控通路；3.构建敲除和转HP1α基因动物模型，验证Dicer-HP1α-MSEs调控RASSF1A启动子从头甲基化机制的正确性。项目主旨是探讨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。

正常细胞恶性转化过程中，以DNA甲基化为代表的表观遗传变化起到重要作用。基因组CpG位点选择性地发生从头甲基化的机制尚不明了，是DNA甲基化的“瓶颈问题”。本项目以Dicer和CyclinD1为研究对象，拟明确胆管癌细胞CyclinD1介导HP1α调控Dicer基因启动子从头甲基化的调控机制。免疫组化染色发现，Dicer和CyclinD1异常表达与胆管癌组织病理特征以及预后不良密切相关。免疫共沉淀（Co-IP）技术表明，CyclinD1特异性募集募集HP1α/H3K9me3/SUV39H1/Dnmts形成复合物。亚硫酸氢盐测序发现，CyclinD1表达与Dicer启动子CpG岛的甲基化水平相关。染色质免疫共沉淀（ChIP）实验表明，CyclinD1募集HP1α/H3K9me3/Suv39h1甲基化复合物于DICER基因启动子区，抑制DICER的表达。双荧光素酶基因报告实验表明，沉默Dicer可特异性下调miRNA促进CyclinD1表达。同时生物学行为实验也显示，敲低CyclinD1能够抑制胆管癌细胞的增殖、侵袭及促进凋亡，联合下调Dicer可部分恢复上述胆管癌细胞的生物学行为。同时我们通过甲基化芯片和蛋白质谱确定了CyclinD1-Dicer作用的关键分子HP1α蛋白下游差异化表达基因。我们的研究表明，CyclinD1特异性偶联HP1α/H3K9me3/Suv39h1/Dnmts复合物抑制Dicer表达，沉默Dicer通过下调miRNA反馈促进CyclinD1表达，从而促进了胆管癌的恶性进展。