动脉粥样硬化血管内皮细胞microRNAs表达谱特征及丹皮酚干预作用研究

The important regulatory role of microRNAs (miRNAs) in cardiovascular disease has been concerned, however, the function and expression profile of vascular endothelial miRNAs in pathogenesis of atherosclerosis (AS) were poorly understood. Screening differentially expressed miRNAs and their target genes in vascular endothelial under pathogenesis of AS are effective way to clarify the relation between miRNAs and AS. Based on the previously research supported by National Science Foundation, the expression profile and expression difference of miRNAs in vascular endothelial with pathogenesis of AS will be screened by the technology of whole genome miRNA chip and verified by real-time quantitative PCR in this project. On these bases, several functional miRNAs will be selected and transient transfected vascular endothelial cells to detect the key molecules in inflammation signaling pathways and their functions through repression or overexpression. Combining with the modern ioinformatics technology, the function of miRNAs and their mechanism will be explored, and target genes or proteins will be evaluated at functional genomics and functional proteomics level. The targets of miRNAs and their mechanisms on AS regulating would be determined and the effect of paeonol would be evaluated. The results of this project would reveal the mechanisms on expression of multi-target genes regulated the biological function of endothelial cell and the scientific connotation of the efficacy of Traditional Chinese Medicine with heat-clearing and detoxifying character, these offer a new strategy for exploring drugs to treat AS.

microRNAs(miRNAs)在心血管疾病中的重要调控作用正引起关注，但目前对动脉粥样硬化(AS)内皮细胞miRNAs的表达及功能知之甚少。寻找AS血管内皮细胞差异性表达的miRNAs以及靶基因、揭示miRNAs与AS关系是有希望的突破口。本课题以已结题国家自然基金研究为基础，采用miRNAs全基因芯片技术筛选AS内皮细胞miRNAs表达谱及表达差异；并用实时荧光定量PCR的方法验证；在此基础上挑选若干个miRNAs通过瞬时转染抑制或过表达检测内皮细胞炎症反应的关键信号分子及其功能变化；结合生物信息学技术探索相应miRNAs执行功能的信号途径，并从功能基因组水平和蛋白水平对候选靶基因/蛋白加以探究。明确miRNAs靶标及其参与调控AS的机制并探讨清热解毒中药丹皮酚的干预作用，揭示AS内皮细胞生物学功能的多靶位基因表达机制和清热解毒中药功效的科学内涵，探索抗AS药物防治新策略。

采用miRNAs全基因芯片技术筛选动脉粥样硬化（AS）血管内皮细胞差异性表达谱miRNAs；并用实时荧光定量PCR的方法验证；在此基础上挑选若干个miRNAs（miR-126、miR-21、miR-30a）通过瞬时转染抑制或过表达检测血管内皮细胞（VECs）炎症反应的关键信号分子及其功能变化。探索相应miRNAs执行功能的信号途径；明确miRNAs靶标及其参与调控AS的机制并探讨清热解毒中药丹皮酚的干预作用，揭示AS内皮细胞生物学功能的多靶位基因表达机制和清热解毒中药功效的科学内涵，探索抗AS药物防治新策略。.① miR-126在ox-LDL诱导的大鼠VECs炎症性反应中具有特异性低表达； VCAM-1为miR-126负调控的靶基因；在ox-LDL诱导的大鼠VECs中，因miR-126表达降低而上调VCAM-1的表达与释放并增强MC与VEC黏附功能。Pae能够促进ox-LDL诱导的VEC 表达miR-126；抑制VCAM-1表达与释放；Pae上调miR-126表达可阻断PI3K/Akt/NF-κB信号通路的转导，降低MC向VECs的黏附。.② ox-LDL可诱导VEC中miR-21的表达。miR-21可下调VECs中PTEN的表达，PTEN是miR-21作用的靶基因。PTEN表达水平的下调，可导致p38 MAPK信号通路的激活，miR-21可能通过激活PTEN/p38 MAPK信号途径介导了ox-LDL诱导的VEC炎症。Pae可通过抑制miR-21的表达，提高靶基因PTEN的表达，抑制p38 MAPK信号通路的激活，减少VEC中炎症因子TNF-α的分泌。.③ 在ox-LDL诱导的大鼠VECs中，miR-30a表达降低，其靶蛋白Beclin-1表达与释放增强，LC3Ⅱ/LC3Ⅰ蛋白表达上升，从而使VECs细胞过度自噬。Pae能够促进ox-LDL诱导的大鼠VECs 表达miR-30a，抑制Beclin-1蛋白表达与释放，向基线水平下调VECs过度自噬以保护VECs。