HO-1基因调控对MDS甲基化的影响及机制研究

Our project team detected the methylation of the MDS related genes' promoter, such as p15INK4B、p16INK4A、DAPK1、CDH1、FHIT, et al. We take the use of lentiviral vector-mediated HO-1 or RNA interference HO-1 combined with the enhanced expression of agent and inhibitor to regulate HO-1 bidirectionally. The methods of Realtime, FISH, Western blot, Flow cytometry, MSP/Gene sequencing, gene chip and animal experiment are also performed in this study. We initially revealed that MDS risk stratification, MDS related genes promoter methylation and demethylating drug sensitivity are related to the expression of HO-1. The effects which the HO-1 gene expression result in MDS related genes promoter hypermethylation occurs are through JAK / STAT, MAPK / ERK, PI3K/AKT pathways to achieve. We will try to explore and confirm these conclusions. Firstly, HO-1 overexpression is a poor prognostic factor in MDS which can be used as an indicator of curative effect evaluation and prognosis evaluation. Secondly, antagonistic or downregulation of the HO-1 gene can enhance the sensitivity of demethylation drugs. Thirdly, using protein chips to verify the above signaling pathways. This project broadens the ideas of MDS pathogenesis studies which provides an experimental basis for MDS targeted therapy and drug development.

本课题组检测MDS患者p15INK4B、p16INK4A、DAPK1、CDH1、FHIT等甲基化相关基因，采用慢病毒载体介导HO-1或si HO-1结合增强剂与抑制剂双向调控HO-1基因的表达，运用Realtime、FISH、Western blot、FCM、MSP/基因测序、基因芯片和动物试验等方法，初步证实了HO-1基因的表达与MDS的危险分层密切相关；与MDS相关基因启动子甲基化有关；与去甲基化药物敏感性有关；HO-1基因表达影响甲基化可能通过JAK/STAT，MAPK/ERK，PI3K/AKT途径实现。试图探索并证实：一、HO-1基因高表达是MDS的不良预后因素，可作为疗效评价和预后评估的参考指标；二、拮抗或下调HO-1基因的表达可增强去甲基化药物的敏感性；三、采用蛋白芯片验证上述信号通路。本项目拓宽了对MDS发病机制探索的思路，为MDS的靶向治疗和药物研发提供了实验依据。

项目背景：骨髓增生异常综合征（Myelodysplastic syndrome，MDS）是起源于造血干细胞的.一组高异质性克隆性疾病。其发病特点为多基因启动子呈过度甲基化，且在 MDS干、祖细胞中发生更频繁，甲基化水平更高，可能是 MDS发生的始动因素之一。去甲基化药物阿扎胞苷或地西他滨为胞嘧啶类似物，能有效抑制 DNMT1使抑癌基因表达升高，使细胞恢复正常的终末分化、衰老或凋亡。然而近年来MDS对去甲基化药物的耐受使得治疗效果不尽人意。探索MDS耐药的机制对提高疗效至关重要。.研究内容：1、MDS患者基因甲基化程度与 HO-1表达相关性的研究；2、HO-1基因表达调控对 MDS-RAEB II细胞株 SKM-1生物学效应及机制的研究；3、体内调控 HO-1基因表达对 DNA甲基化影响及机制研究。.重要结果及关键数据：1、小干扰RNA沉默HO-1增强低浓度5-氮杂胞苷诱导SKM-1细胞凋亡，HO-1高表达可以增加DNMT3对p16基因启动子的甲基化作用；2、抑制HO-1增加地西他滨对骨髓增生异常综合症（MDS）细胞中p15基因去甲基化作用；3、抑制HO-1表达通过减弱DNMT1的活性，增强地西他滨对p15的去甲基化作用。.科学意义：为临床治疗MDS逆转去甲基化药物耐药提供理论依据。