缺氧条件下miR145负调控CaMKⅡ对血管平滑肌细胞自噬作用及生物学效应的影响

It has been shown that hypoxia in vascular wall occurs during atherosclerosis. In this process, vascular smooth muscle cells tend to proliferate and migrate. The expression of miR-145 is dramatically suppressed in smooth muscle cells. However, the underlining mechanisms remain unknown. We found that hypoxia significantly inhibited the miR-145 expression, which negatively regulated HIF-1 alpha expression and promoted the autophagy of smooth muscle cells. In this study, we aim to confirm that miR-145 could negatively regulated CaMKII and HIF-1 alpha expression in hypoxia conditionusing gene transfection, confocal microscopy and electron microscopy analysis. Meanwhile, miR-145 could promote autophagy of VSMC and stimulate the secretion some factors through PI3K/Akt/mTORC-1 pathway, which cause the phenotype transformation, proliferation and migration of VSMC. In miR-145 knockout atherosclerosis mice model, we will use 20% Pluronic hydrogel containing miR-145 expression plasmid to coat balloon and injury aortic. To test the possibility of miR-145 as a gene therapy target for atherosclerosis, , we will use echocardiography and other methods to assess local stenosis. The expression level of miR-145 will be detected in collected blood from coronary angiography patients, and the correlation between miR-145 and severity of coronary artery disease will be studied. The results will provide the experimental and theoretical basis for miR-145 as a new target on atherosclerosis diagnosis and treatment.

研究显示动脉粥样硬化（AS）发生时血管壁出现缺氧，血管平滑肌细胞发生增殖与迁移，而在平滑肌细胞中高表达的miR-145被明显抑制。我们在研究中发现缺氧明显抑制miR-145的表达，且可以负调控CaMK II，具体机制及生物学效应值得研究。本课题拟通过基因转染、共聚焦显微镜、电子显微镜等技术，验证缺氧时miR-145负调控其靶基因CaMKⅡ，影响HIF-1α的表达，并通过影响PI3K/Akt/mTORC-1途径，促进VSMC自噬作用，从而引起VSMC的表型转化、增殖及迁移；采用miR-145基因敲除小鼠AS模型，利用含有miR-145慢病毒的20% Pluronic水凝胶涂层球囊损伤主动脉，通过超声心动图等方法评估局部管腔狭窄程度；采集行冠脉造影患者血浆检测miR-145表达水平，研究其与冠脉病变程度的相关性。研究结果为miR-145作为AS诊断及治疗新靶点提供实验和理论依据。

研究目的：动脉粥样硬化（AS）发生时血管壁出现缺氧，血管平滑肌细胞发生增殖与迁移，而在平滑肌细胞中高表达的miR-145被明显抑制。我们在研究中发现缺氧明显抑制miR-145的表达，且可以负调控CaMK II，可能在粥样动脉硬化的形成和进展中发挥重要作用，具体机制及生物学效应值得研究。方法：本课题在采集行冠脉造影患者血浆检测miR-145表达水平，通过qPCR等技术测定血样中miR145水平，研究结果表明血中mir-145与冠心病有显著相关性的相关性。在基础实验方面，选取小鼠原代血管平滑肌细胞（VSMC）进行细胞培养，分别以低氧浓度（3%）和正常氧浓度（21%）干预细胞培养，本研究通过转染miR-145基因转染、western blot等技术，验证了缺氧时miR-145负调控其靶基因CaMKⅡ，通过beclin1蛋白调控，促进VSMC自噬作用;并用免疫荧光法检测经过转染干预的细胞中LC3的荧光表达水平；采用动脉球囊损伤制造小鼠AS模型，成功造模；结果：1、研究结果表明血中mir-145与冠心病有显著相关性的相关性。2、在缺氧条件下，血管平滑肌细胞中mir-145的表达水平降低，而CaMKⅡ、beclin1表达水平升高。3、小鼠造模结果提示结论，球囊损伤部位造成了血管中更严重的斑块和狭窄：缺氧条件下，细胞内mir-145表达下调，并且是以经CaMKⅡ/beclin1途径来进行调节的。因此，缺氧可以诱导斑块中的平滑肌细胞生存进而促进斑块进展，从而加重粥样斑块以及动脉粥样硬化的发展。作为AS诊断及治疗新靶点提供实验和理论依据。