MiRNA expression profile in murine advanced atherosclerotic plaque compared with un-diseased arterial wall

Aberrantly expressed miRNAs contribute to developmental abnormalities and diseases such as cancer, diabetes, and cardiovascular disorders, by hybridizing to specific mRNA targets and repressing their translation into proteins. Although miRNA expression signature is characterized in the process of neointimal thickening during proliferative vascular diseases such as atherosclerosis, so far global miRNA expression profiling in the different stages of atherosclerosis is completely unknown. We explored stage-specific microRNA signatures in the progress of atherosclerosis in hyperlipidemia mouse model, which may help to identify the critical miRNAs contributing atherosclerotic development and stabilization. Female apoe-/- mice (6-8 weeks, Jackson Laboratory) were fed a high fat diet (HFD, 21% crude fat, 0.15% cholesterol and 19.5% casein, Altromin, Germany) for 3 or 10 months (N=3-4). Serial sections (20 µm thick) of aortic roots and carotid arteries from these mice were mounted on membrane mounted metal frame slides (MMI), deparaffinized under RNase-free conditions, and completely dried. Laser capture microdissection (LCM) was performed with a laser microdissection system (MMI cellcut plus, Molecular Machines and Industries, Switzerland) assembled onto an inverted microscope (Olympus IX71). Plaque tissue or morphologically normal vessel wall of at least 40 sections per mouse were collected. RNA was isolated using RecoverAll total nucleic acid isolation kit (Applied Biosystems) according to the manufacturer’s instructions.

异常表达的微小RNA（microRNA, miRNA）可通过与特定信使RNA（messenger RNA, mRNA）靶标杂交并抑制其翻译为蛋白质，参与发育异常及癌症、糖尿病、心血管疾病等多种疾病的发生发展。尽管已有研究在动脉粥样硬化等增殖性血管疾病的内膜增厚过程中解析了miRNA的表达特征，但截至目前，动脉粥样硬化不同阶段的全基因组miRNA表达谱仍完全未知。本研究针对高脂血症小鼠模型的动脉粥样硬化进展过程，探究了其阶段特异性的miRNA表达特征，以期助力识别参与动脉粥样硬化发生发展与斑块稳定的关键miRNA。选取来自杰克逊实验室（Jackson Laboratory）的6~8周龄载脂蛋白E基因敲除雌性小鼠（apoe-/-小鼠），给予高脂饲料（high fat diet, HFD），其成分为21%粗脂肪、0.15%胆固醇及19.5%酪蛋白，购自德国Altromin公司，分别饲喂3个月或10个月，每组动物数为3~4只（N=3~4）。将这些小鼠的主动脉根部与颈动脉制备为厚度20 μm的连续切片，粘贴于带膜金属框玻片（membrane mounted metal frame slides, MMI）上，在无核糖核酸酶（ribonuclease, RNase）环境中进行脱蜡处理后完全干燥。使用搭载于奥林巴斯IX71倒置显微镜（Olympus IX71）的激光显微切割系统（MMI CellCut Plus，瑞士Molecular Machines and Industries公司）完成激光捕获显微切割（laser capture microdissection, LCM）操作。每只小鼠至少收集40张切片对应的斑块组织或形态学正常的血管壁组织。按照试剂盒说明书的操作流程，使用RecoverAll总核酸分离试剂盒（Applied Biosystems公司）提取RNA。