miR-320a作为冠心病标志物的临床价值及其参与动脉粥样硬化形成的机制研究

Atherosclerosis cardiovascular disease was high morbidity and mortality, which mechanism was still not understood. Our previous study found that the serum miR-320a in patients with coronary heart disease and miR-320a in the foam cell formation were abnormal expressed, which were related with blood lipids and the severity of coronary artery. Bioinformatics analysis revealed that there were transcription factors at upstream of miR-320a. And miR-320a could targeted regulation downstream ABCG1 and LPL, however, the mechanism was not clear. Thus, we hypothesized that miR-320a is potential molecular biomarkers of coronary heart disease, and transcription factor could regulate expression of miR-320a, then miR-320a adjusts ABCG1 and LPL expression, to influence intracellular cholesterol accumulation, which involved in the development of atherosclerosis. In order to verify the hypothesis, we will adopted clinical samples, foam cells and atherosclerosis mouse models and use some technique such as qRT-PCR, lentivirus transfection, transcription factor chip, etc. to evaluate the clinical value of miR-320a for coronary heart disease. We will screen and verify the transcription factor of miR-320a, and clear the relationship among miR-320a, ABCG1 and LPL. Our study will also contruct the regulatory pathway between miR-320a and its upstream transcription factors and downstream target genes, and explore the relationship between this pathway and the progression of atherosclerosis. The purpose of our study is to provide experimental and theoretical foundation for further elucidating the mechanism of the development of atherosclerosis.

动脉粥样硬化性（As）心血管疾病发病率、死亡率高，机制未完全阐明。我们前期研究发现冠心病患者血清中、泡沫细胞形成中miR-320a表达异常，且与血脂和冠脉严重程度相关；生物信息学显示miR-320a上游存在转录因子，下游可靶向调控ABCG1和LPL，机制尚不明确。据此提出假说：miR-320a是冠心病潜在标志物，在转录因子的调控下调节ABCG1和LPL的表达，影响细胞内胆固醇的聚集，参与As的发生发展。为验证假说，本项目拟通过临床标本、泡沫细胞和As小鼠模型，采用qPCR、慢病毒转染、转录因子芯片等技术，评估miR-320a对冠心病的临床价值；筛选和验证调控miR-320a表达的转录因子，明确miR-320a与ABCG1和LPL之间的关系，构建miR-320a与其上游转录因子和下游靶基因所形成的调控通路，探讨该通路与As形成的联系，为进一步阐明AS的发生发展机制提供实验基础和理论依据。

课题组早期的低密度芯片结果显示冠心病患者血清miR-320a水平上调，但miR-320a在动脉粥样硬化性心血管疾病（CVD）患者血清中表达的临床价值尚未评估，其参与动脉粥样硬化过程的作用机制尚不清楚。为此，本项目首先采用 qRT-PCR技术对患者血清miR-320a表达变化的基因芯片结果进行验证，分析其与血脂和炎症指标的关系。结果显示急性冠脉综合征（ACS）和稳定性冠心病（SCAD）患者血清中miR-320a表达量均显著升高，血清miR-320a与ox-LDL、HDL-C、冠脉最大狭窄程度和病变支数密切相关，提示miR-320a可能通过调节血脂水平，参与As的发生发展。体外细胞实验中，通过生物信息学软件对miR-320a的靶基因进行预测，采用Western Blot技术和荧光素酶报告实验对于预测结果进行验证；采用ELISA、高效液相、酶法等实验检测miR-320a对于泡沫细胞内胆固醇外流、胞内胆固醇酯构成及细胞因子分泌的影响。结果显示，ox-LDL引起泡沫细胞内miR-320a水平增高，miR-320a能通过靶向抑制ABCG1的表达，减少泡沫细胞胆固醇外流、增加炎症因子分泌并增加细胞内胆固醇酯含量，最终促进泡沫细胞形成。体内实验中，通过慢病毒载体在高脂诱导AS模型小鼠体内实现了miR-320a的过表达或抑制。结果显示，过表达miR-320a可促进动脉粥样硬化的发生发展。结合临床研究、体外细胞研究和体内动物研究结果，本项目证实miR-320a具备作为AS性CVD分子标志物潜能，能通过抑制ABCG1的表达促进泡沫细胞炎症因子的分泌，降低胆固醇外流，增加泡沫细胞内胆固醇酯含量，从而加速AS进程。依托本项目累计发表论文18篇，其中SCI论文8篇，多次参加学术会议，可为AS性CVD的诊断、治疗及发病机制研究奠定理论基础。