SMAD3基因启动子DNA甲基化及其下游miRNA水平联合检测作为冠心病新型分子标志物的临床价值及作用机制

DNA methylation and circulating microRNA may function as the novel molecular biomarkers for cardiovascular diseases (CVD). However, their clinical values of joint-detection and cooperative molecular mechanisms involved in the pathogenesis and progression of CVD remain unknown. Our recent studies found that SMAD3 promoter methylation incidences were significantly decreased while its downstream miR-548h levels were significantly increased. Further bioinformatics analyses and cell experiments demonstrated that both SMAD3 and miR-548h may participate in the regulation of atherosclerosis-associated signaling pathways. Thus, we hypothesize that the joint-detection of SMAD3 promoter methylation and its downstream miR-548h may be served as the novel indicators for the diagnostic typing, condition monitoring and prognostic assessing of CVD patients. This novel joint-detection pattern may improve diagnostic accuracy and rightly reflect the severity and progression of atherosclerotic lesions. Both SMAD3 and miR-548h may be implicated in origin and development of atherosclerosis via their cooperative regulation of atherosclerosis-associated cell functions, and may subsequently act as the potential therapeutic targets for CVD. In order to verify the hypothesis, we perform a series of experiments in clinical specimens and cells lines to fully definite the clinical values of jointly detecting SMAD3 promoter methylation and its downstream miR-548h for CVD, and their cooperative molecular mechanisms involved in atherosclerosis. We suspect that the results from this project will provide novel approaches and theoretical bases for the diagnoses and therapies of atherosclerotic CVD.

DNA甲基化和循环microRNA有望成为心血管疾病（CVD）新型分子标志物，但二者联合检测价值及共同参与CVD发生发展分子机制尙不清楚。我们最新研究显示，冠心病患者SMAD3启动子甲基化水平显著降低，而其下游共表达的miR-548h水平显著上调；生物信息学分析与细胞实验显示二者可共同参与动脉粥样硬化（As）相关信号通路调节。据此提出假说：SMAD3基因启动子甲基化及其下游miR-548h联合检测是CVD患者诊断分型、病情监控及预后评估新指标，二者联合检测可提高疾病诊断精确性、准确反映病变严重程度和病情进展；SMAD3和miR-548h可共同调控As相关细胞功能参与As发生发展，是CVD潜在治疗靶点。为验证假说，本项目拟从临床样本、细胞水平全面探讨SMAD3基因启动子甲基化及其下游miR-548h对CVD的临床价值；明确二者协同参与As机制，为As性CVD的诊治提供新的途径和理论依据。

DNA甲基化和循环microRNA有望成为心血管疾病（CVD）新型分子标志物，但二者联合检测价值及共同参与CVD发生发展分子机制尙不清楚。本项目临床研究纳入多中心队列的急性冠脉综合征（ACS）、稳定性冠心病患者（SCAD）和健康体检对照者作为研究对象，先后通过焦磷酸测序、实时定量甲基化特异性PCR（RQ-MSP）技术分别检测其外周血单个核细胞（PBMCs）SMAD3、DNM1等基因特定CpG位点甲基化水平，通过实时定量PCR（RT-qPCR）技术检测血浆miR-199b-5p等相关microRNA水平，发现联合检测SMAD3、DNM1等基因特定CpG位点甲基化水平及miR-199b-5p等相关microRNA水平，可作为ACS、SCAD患者液体活检分子标志物的新组合，比单一指标更具价值；机制研究建立人脐静脉内皮细胞（HUVEC）损伤模型，通过焦磷酸测序和甲基化酶抑制剂处理等实验，发现位于DNM1基因内部的miR-199b-5p启动子区甲基化程度降低可能是导致HUVEC损伤模型中miR-199b-5p表达上调的原因；进一步通过靶基因预测、miR-199b-5p mimic转染、荧光素酶报告基因检测、蛋白免疫印迹（WB）等手段，发现miR-199b-5p可通过靶向MLK3来调控HUVEC细胞的黏附功能，从而参与AS发生、发展进程。本项目累计已发表论文21篇，其中SCI收录6篇；获得科研奖励1项，授权国家发明专利2项，举办继续教育培训班2次；培养博士、硕士研究生9名，博士后3名，为ASCVD的诊断、治疗及发病机制研究提供了新的依据。