miR-17调控VEGF对糖尿病心肌梗死后心肌微血管新生的作用及其机制研究

Angiogenesis has great effect on the remodeling and prognosis post myocardial infarction. Our recent study showed that VEGF production mediated by PKCβ2 and VEGF-dependent Akt/eNOS signaling pathways which concerns about post-myocardial infarction of diabetes mellitus, the average relative activity of miR-17 rises post ischemia in diabetic C57BL/6 mice with negative correlation to VEGF expressions, the VEGFAa protein expression level of HUVECs increases when challenged with miR-17 inhibitor. So, we infer that miR-17 targeting VEGF regulates angiogenesis in high glucose and has effect on remodeling post myocardial infarction in diabetes mellitus. To certify whether miR-17 targeting VEGF regulates angiogenesis, we design luciferase reporter gene vector of 3’UTR of VEGF mRNA firstly and transfect to HUVECs challenged with miR-17 mimic or control respectively. At the same time, miR-17 over-expression/inhibitor adenovirus will be used to interfere with the expression of miR-17 of the primary myocardial microvascular endothelial cells to make clear the role of miR-17 targeting VEGF which regulates angiogenesis. Finally, we will demonstrate that miR-17 targeting VEGF regulates angiogenesis on diabetic/normal C57BL/6 mice with miR-17 antagomirs/agomirs in vivo meanwhile with cardiac specific miR-17 knock out and VEGF mutant transgenosis mice. In conclusion，we would reveal the underlying mechanism of miR-17 which targets VEGF on angiogenesis in high glucose and try to provide a novel therapeutic target for promoting angiogenesis and modifying myocardial remodeling post myocardial infarction in diabetes mellitus.

心肌梗死后心肌组织微血管新生对心肌修复及预后产生重要影响，而糖尿病心肌梗死后心肌微血管新生不良。我们的预实验结果提示糖尿病心肌梗死后心肌微血管新生受PKCβ2介导的VEGF生成及Akt/eNOS信号通路调控；在人脐带静脉血内皮细胞中抑制miR-17表达后VEGFAa蛋白表达显著升高。故我们提出假说：miR-17调控的VEGF表达在糖尿病心肌梗死后心肌微血管新生机制中发挥重要作用。我们拟在体外进一步确定miR-17对VEGF的调控作用基础上，以C57BL/6小鼠原代心肌微血管内皮细胞为研究对象，通过调节miR-17的表达研究在高血糖条件下miR-17调控的VEGF表达对微血管形成的作用和上游调节机制，并分别构建心脏特异性miR-17条件敲除和VEGF突变体转基因小鼠模型，研究miR-17表达对糖尿病心梗后心脏微血管新生及心肌重构的影响及调控机制，为改善糖尿病心肌梗死的预后提供新思路。

有报道显示miR-17参与了多种肿瘤血管内皮细胞的增殖、迁移及血管新生的调控。我们在前期预实验中发现：糖尿病C57BL/6小鼠心肌梗死后miR-17表达显著上调，其水平与VEGF的生成负相关； miR-17抑制后HUVECs内VEGFAa蛋白表达水平显著升高，提示miR-17可能通过调控VEGF影响高糖环境下血管形成。因此，我们推测通过调节miR-17可改善高糖环境下微血管新生，进而促进糖尿病C57BL/6小鼠心肌梗死后修复、改善心肌重构，其机制可能和miR-17靶向调控VEGF有关。针对VEGF mRNA 3’-UTR的miR-17结合位点构建双荧光素酶野生型和突变型质粒载体，与mi-17 mimic和mi-17 mimic negative control (mimic NC)共转染后检测荧光素酶的活性变化。发现与mimic NC相比，在VEGF mRNA 3’-UTR野生型中转染mi-17 mimic后荧光素酶活性显著下降，而VEGF mRNA 3’-UTR突变型转染mi-17 mimic后荧光素酶活性无明显变化。培养脐静脉内皮细胞（HUVEC），细胞传代后分为对照组、miRNA过表达阴性对照组（转染miR-17 mimic NC）、miRNA降表达阴性对照（转染miR-17 inhibitor NC）组，miR-17过表达组（转染miR-17 mimic）和miR-17降表达组（转染miR-17 inhibitor）, 检测VEGF的表达，PCR检测VEGF的表达发现与阴性对照组相比，转染miR-17 mimic使VEGF蛋白表达明显下调，而VEGF mRNA表达水平无明显差异。转染miR-17 inhibitor后VEGF蛋白表达明显上调，而VEGF mRNA表达水平均无明显差异。通过脐静脉内皮细胞（HUVEC），研究高血糖环境下miR-17对心肌微血管内皮细胞迁移及成管功能的影响，及其与VEGF的关系。发现与正常糖组相比，高糖组细胞的增殖，迁移和管腔形成能力明显降低，miR-17水平增加而VEGF的表达减少。抑制miR-17明显改善高糖条件下的细胞迁移和管腔形成能力，同时增加VEGF的表达。最后在体动物水平进行验证，得出了糖尿病心肌梗死后，miR-17表达增加，抑制miR-17的水平可以通过增加血管生成从而改善心脏重构和心功能。