LncRNA H19调控miR-22促进冠脉旁路移植术后血管新生的信号传导机制

    Signal conduction mechanism of LncRNA H19 in promoting angiogenesis after coronary artery bypass grafting via regulating miR-22

      摘要
    • 摘要:
      目的 构建冠脉旁路移植术后大鼠模型，评估LncRNA H19通过miR-22促进缺血心肌组织血管新生的信号传导机制。
      方法 选择SD大鼠20只，随机分为2组：观察组，予以结扎冠脉前降支行自体右颈外静脉—颈总动脉移植术建立冠脉搭桥术模型；对照组，予以假手术处理。于术后第2周处死大鼠，取大鼠心脏，培养心肌微血管内皮细胞（CMEC）。qRT-PCR检测并比较2组CMEC中LncRNA H19和miR-22的表达水平；构建shRNA H19干扰LncRNA H19的表达。通过脂质体Lipfectamine 2000介导将miR-22 mimics及其NC、inhibitor-miR-22及其NC转染至CMEC细胞，应用Transwell法检测CMEC细胞的迁移能力；应用MTT增殖试验检测CMEC细胞增殖活性；应用Matrigel matrix方法测定内皮细胞成管能力。
      结果 与对照组比较，观察组LncRNA H19与miR-22呈高表达（P<0.05，P<0.01），且LncRNA H19表达水平与miR-22表达水平呈正相关（r=0.772，P<0.05）。shRNA H19能有效干扰观察组CMEC细胞的miR-22表达（P<0.05）。miR-22-mimics组CMEC细胞迁移能力最高，miR-22-inhibitor组最低（P<0.05），而miR-22 NC组、miR-22-mimicsNC组与miR-22-inhibitor NC组无差异。MTT细胞增殖试验显示，5组CMEC细胞OD值存在显著差异，miR-22-mimics组CMEC细胞最高，miR-22-inhibitor组最低（P<0.05），而其他3组无差异。miR-22-mimics组CMEC细胞成管能力最高，miR-22-inhibitor组最低（P<0.05），而其他3组无差异。
      结论 LncRNA H19能通过上调miR-22表达水平形成信号传导通路，从而促进冠脉旁路移植术后大鼠新生血管生成。

       

      Abstract:
      AIM To construct a rat model of coronary artery bypass transplantation and to evaluate the signaling mechanism of LncRNA H19 in promoting angiogenesis in ischemic myocardial tissue via regulating miR-22.
      METHODS Twenty SD rats were selected and randomly divided into 2 groups: observation group (treated by ligation of the anterior descending artery and the right external jugular vein-common carotid artery graft) and control group (treated with sham surgery). All rats were killed in the second week after surgery, hearts were harvested and myocardial microvascular endothelial cells (CMEC) were cultured. The expression levels of LncRNA H19 and miR-22 were measured by qRT-PCR and compared between the two groups. shRNA H19 was constructed and interfered to the expression of LncRNA H19. The miR-22 mimics and NC, and inhibitor-miR-22 and NC were transfected to CMECs induced by liposomal (Lipfectamine 2000), respectively. The invasion ability of the four CMECs groups was detected with Transwell assay, the cell proliferation ability of the five CMECs groups was detected with MTT and the tube forming capacity of CMECs was determined by Matrigel matrix.
      RESULTS The expression levels of LncRNA H19 and miR-22 in the observation group were higher than those in the control group (P<0.05, P<0.01). The positive relationship of LncRNA H19 expression levels and miR-22 expression levels was confirmed (r=0.772, P<0.05). shRNA H19 could effectively interfere with the expression of miR-22 in CMECs of the observation group (P<0.05). The migration ability of CMECs by miR-22-mimics was the highest and by miR-22-inhibitor was the lowest (P<0.05), while miR-22-NC, miR-22-mimics NC and miR-22-inhibitor NC showed no difference. MTT cell proliferation test showed significant differences in OD values between the five groups (P<0.05). The OD values of CMECs by miR-22-mimics was the highest and by miR-22-inhibitor was the lowest (P<0.05), while the other 3 CMEC groups showed no difference. The tube formation ability of CMEC by miR-22-mimics was the highest and by miR-22-inhibitor was the lowest (P<0.05), while the other 3 CMEC groups showed no difference.
      CONCLUSION LncRNA H19 up-regulates the expression level of miR-22 to form a signaling axis and thus promotes the new angiogenesis in rats after coronary artery bypass grafting.

       

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