摘要
目的 观察动脉粥样硬化患者血清中微小RNA-181b（miR-181b）的表达情况,以及miR-181b对人血管内皮细胞核内转移蛋白-α3（Importin-α3）表达和血管平滑肌细胞增殖、迁移的影响,探讨miR-181b在动脉粥样硬化发生发展过程中的作用.方法 选取2012年11月至2013年6月在上海交通大学医学院附属第三人民医院就诊的经外周动脉超声检查诊断为动脉粥样斑块形成的50例患者为动脉粥样硬化组,平均年龄（78.1±8.9）岁;同时入选50名健康成人为对照组,平均年龄（72.5±10.7）岁.采集患者外周血,采用带茎环结构的实时定量PCR方法检测其血清中miR-181b 的表达水平;经Targetscan和Pitar靶基因预测数据库预测核质转运受体Importin-α3为miR-181b的靶基因,体外实验采用Western blot检测miR-181b对人血管内皮细胞Importin-α3表达的影响;采用双荧光素酶报告基因检测证实Importin-α3为miR-181b的直接靶基因;CCK8法和Transwell小室试验检测miR-181b对人血管平滑肌细胞增殖、迁移能力的影响.结果 与正常组比较,动脉粥样硬化组血清中miR-181b的表达水平较低（31.69±0.96比82.28±5.95,P＜0.05）;Western-blot和双荧光素酶报告实验证实Importin-α3为miR-181b的直接靶基因;增高miR-181b的表达使得人血管平滑肌细胞的增殖（1.57±0.18比2.66±0.16,P＜0.05）和迁移能力（8.7±1.1比21.4±2.3,P＜0.05）明显降低;抑制miR-181b的表达则获得相反的效果（增殖：2.88±0.09比2.04±0.11,P ＜0.05;迁移：15.2±1.5比8.4±1.3,P＜0.05）.结论 动脉粥样硬化患者血清中miR-181b表达水平低于正常人,miR-181b可能通过阻断血管内皮细胞中核因子-κB信号途径和通过抑制血管平滑肌细胞的增殖和迁移起到抗动脉粥样硬化的作用.
Objective To observe the serum expression of miR-181b in atherosclerotic patients and the in vitro effects of miR-181b on vascular smooth muscle cell growth and migration.Methods Fifty patients （mean age：（78.1 ± 8.9） years old） with carotid ultrasound examination evidenced atherosclerotic plaque were enrolled as the atherosclerosis group and 50 healthy （mean age：（72.5 ± 10.7） years old） subjects serve as control group.Stem-loop real time RT-PCR was used to detect the serum expression of miR-181b.Importin-α3 was predicted to be a direct target of miR-181b by Targetscan and Pictar.Western-blot was employed to detect the in vitro effects of miR-181b on the expression of Importin-α3 in endothelial cells.Luciferase reporter assay was employed to testify the prediction.The effects of miR-181b on vascular smooth muscle cell growth,migration abilities were respectively examined by CCK8 assay and Matrigel migration assay.Results Compared with healthy controls,serum expression of miR-181b was significantly downregulated in patients with atherosclerosis （31.69 ± 0.96 vs.82.28 ± 5.95,P 〈 0.05）;Importin-α3 was predicted and proved to be a direct target of miR-181b by Western-blot and luciferase reporter assay.The proliferation and migration of vascular smooth muscle cell were significantly downregulated by forced expression of miR-181 b （1.57 ± 0.18 vs.2.66 ± 0.16,P 〈 0.05;8.7 ± 1.1 vs.21.4 ± 2.3,P 〈 0.05）,while these effects could be abolished by inhibition of miR-181b（2.88 ±0.09 vs.2.04 ±0.11,P 〈0.05;15.2 ± 1.5 vs.8.4 ± 1.3,P 〈 0.05）.Conclusion The serum miR-181 b level was significantly reduced in patients with atherosclerosis.miR-181b may function as an atherosclerosis suppressor by interupting the NF-κB pathway in endothelial cells and inhibiting the proliferation and migration of vascular smooth muscle cells.
出处
《中华心血管病杂志》
CAS
CSCD
北大核心
2015年第6期516-520,共5页
Chinese Journal of Cardiology
