组蛋白乙酰化和miRNA介导的肾癌SLC22A2低表达的机理及应用研究

Organic cation transporter OCT2 encoded by the SLC22A2 gene is highly expressed in renal tissue and translocates platinum-like anticancer drugs into renal cells. Kidney cancer is highly resistant to chemotherapy. Our previous studies have shown that SLC22A2 is low in renal cell carcinoma and its DNA methylation plays an important role（Sci Transl Med,2016）. On the basis of this, this project further revealed tht histone acetylation H3K18Ac/K27Ac and miR-630/miR-489-3p involved in the regulation of SLC22A2 gene transcriptional regulation in renal cell carcinoma, and the relationship between histone acetylation or miRNA and DNA methylation of SLC22A2 in renal cell carcinoma. The combined administration of epigenetic drugs with OCT2 substrate oxaliplatin in the treatment of RCC in vitro and in vivo is designed using RCC cells, RCC cell xenograft nude mice and PDX models and the pharmacological mechanism and toxicity are studied. This study will enrich the SLC22A2 regulatory pathway in RCC, improve the epigenetic regulation mechanism of SLC22A2, and provide theoretical and experimental basis for clinical diagnosis and treatment of RCC.

有机阳离子转运体OCT2由SLC22A2基因编码，在肾组织中高表达，转运铂类抗癌药物进入肾细胞，而肾癌对化疗广泛耐药。我们的研究表明，在肾癌组织中SLC22A2低表达，其DNA甲基化发挥了重要作用（Sci Transl Med,2016）。在此基础上，本项目进一步揭示SLC22A2组蛋白乙酰化修饰H3K18Ac/K27Ac和miR-630/miR-489-3p参与肾癌SLC22A2基因转录调节的机理，探究肾癌中SLC22A2组蛋白乙酰化或miRNA与DNA甲基化之间的关系；并基于肾癌中SLC22A2的表观遗传学机理，从RCC细胞、RCC细胞异种移植瘤裸鼠和PDX模型的体内外实验，设计表观遗传药物与OCT2底物奥沙利铂治疗RCC的联合给药方案，并验证其有效性和安全性。本研究将丰富RCC中SLC22A2基因调节信号通路,完善SLC22A2基因表观遗传调控机理，为临床诊断和治疗RCC提供依据。

本项目主要研究成果如下：.一、阐明了表观遗传调控RCC中的OCT2的信号通路：转录因子MYC可以作为DNA甲基化与组蛋白修饰之间的纽带，揭示DNA高甲基化阻遏MYC与组蛋白甲基化信号（H3K4me3）和组蛋白乙酰化信号（HDAC）的相关蛋白的相互作用，DAC使OCT2启动子区去甲基化从而增加MYC与OCT2启动子区E box位点的结合，促进MYC招募MLL1和HDAC7/9。MLL1催化OCT2启动子区H3K4me3，提高OCT2基因转录水平；HDAC7/9是负责催化OCT2启动子区的组蛋白去乙酰化酶，合用SAHA抑制富集的HDAC7/9，协同上调OCT2基因的转录。.二、揭示了miR-630和miR-489-3p与OCT2的作用机制：这两种miR通过直接结合到OCT2的3‵-UTR抑制其表达，并在体外和异种移植瘤中减少摄取奥沙利铂。与癌旁组织相比，miR在RCC中显著上调，与OCT2表达水平呈负相关。RCC中c-Myc与pri-miR-630启动子的结合增加，引起miR-630的上调。总之，这两种miR通过直接靶向RCC中的OCT2发挥阻碍肿瘤治疗的作用；RCC治疗中合用miR拮抗剂在可使细胞对奥沙利铂敏感，为临床化疗提供依据。.三、发现lncRNA SANT1调节RCC中SLC47A2表达的机制：c-Myc/E2F1/miR-20a环路通过c-Myc/MLL1和E2F1/HDAC1表观调节复合物参与SLC47A2启动子区H3K4me3和H3K27ac修饰,表现出对SLC47A2的双向调节机制；SANT1可特异结合于SLC47A2的表达抑制复合物SFPQ/E2F1/HDAC1，其完整的高级结构是形成RNA-蛋白复合物并使E2F1/HDAC1脱靶的基础；SANT1的功能与c-Myc/E2F1/miR-20a环路密切相关，是SLC47A2表达调节网络的重要参与者。