冠心病血瘀证相关miRNA表达的甲基化调控机制研究

Coronary heart disease (CHD) is a complex polygenic disease result from both genes and environment. Blood stasis is one of the most common TCM syndromes of coronary heart disease and its pathogenesis involves different levels of biological networks such as epigenetic regulation. Prior research has shown that epigenetic factors such as DNA methylation, microRNAs involved in the formation of TCM syndrome of coronary heart disease. The expression level of methylation miRNA can cause serious pathological consequences. The promoting blood circulation and removing blood stasis decoction to treat CHD may be by changing the CpG island methylation level in promoter regions of microRNAs, restoring the balance of microRNAs and its target genes, keeping the dynamic balance of body internal environment. This study intends to choose cases of clinical confirmed CHD by coronary angiography with typical blood stasis syndrome. Using the miRNA expression profile chip, methylation chip, cloning sequencing and bioinformatics analysis and qRT-PCR technology, this study will screen and validate DNA methylation regulation miRNAs related with CHD and blood stasis syndrome and verified in vitro. At the same time using typical promoting blood circulation drug Xuesetong capsule to intervene, this study will clarify the scientific connotation of syndrome combined disease and formulas combined syndrome and provide new targets for the effective drug to treat CHD with blood stasis syndrome.

冠心病是多基因复杂疾病，是基因和环境共同的作用结果，血瘀证是冠心病最常见的证型之一，其发病机理涉及表观遗传等不同层次生物网络调控。前期研究表明，DNA甲基化，microRNA等表观遗传因素参与了冠心病中医证候表型的形成，受甲基化调控的miRNA表达量增加或减少均能引发严重的病理后果。活血化瘀方药可能是通过改变miRNA启动子区域CpG岛甲基化水平，恢复相关miRNA与靶基因之间的消长平衡，使机体内环境保持动态平衡来治疗冠心病血瘀证。本研究拟选择临床经冠脉造影确诊的冠心病血瘀证典型病例，利用miRNA表达谱芯片、甲基化芯片、克隆测序、生物信息学分析和qRT-PCR等技术，筛选并验证受DNA甲基化调控的冠心病血瘀证相关microRNA并进行体外验证。同时用活血化瘀代表方药血塞通胶囊进行干预，阐述冠心病血瘀证病证结合、方证相应理论的科学内涵，为研制治疗冠心病血瘀证的有效方药提供新的靶标。

本冠心病是多基因复杂疾病，是基因和环境共同的作用结果，血瘀证是冠心病最常见的证型之一，其发病机理涉及表观遗传等不同层次生物网络调控。本课题严格按照项目要求完成了以下工作：①选择临床经冠脉造影确诊的冠心病血瘀证典型病例，利用miRNA表达谱测序、甲基化测序、焦磷酸测序、甲基化PCR、生物信息学分析和qRT-PCR等技术，筛选并验证受DNA甲基化调控的冠心病血瘀证相关microRNA及其靶基因；②利用miR194转染、去甲基化处理HUVEC细胞进行体外验证，明确冠心病血瘀证特异DNA甲基化-miRNA-靶基因信号通路的作用关系；③活血化瘀代表方药血塞通胶囊进行干预，以药测证，观察相关基因的变化，明确冠心病血瘀证变化与相关基因通路的关系。本研究发现：①冠心病血瘀证中存在DNA甲基化位点、miRNA和mRNA的差异基因表达谱，包括28461个甲基化位点、295个miRNA和470个mRNA，基因功能涉及免疫和炎症反应通路，存在冠心病血瘀证特异的DNA甲基化-miRNA-mRNA调控网络，并筛选出miR194 promoter-miR194-MAPK关键信号通路。研究提示，此信号通路与冠心病血瘀证密切相关，有成为冠心病血瘀证生物标志物的可能；②通过细胞实验，确定了miR194启动子甲基化-miR194-MAPK依次调控的关系；③通过临床RCT试验，发现miR194 promoter-miR194-MAPK信号通路的关键节点明显改变，提示此信号通路不仅与冠心病血瘀证密切相关，还有可能成为冠心病血瘀证的治疗靶标。由此发现，miR194 promoter-miR194-MAPK信号通路很有可能成为冠心病血瘀证的生物标志物和治疗靶标。此研究揭示了冠心病血瘀证的物质基础和科学内涵，并对诊断生物标志物提供了新的思路。