MicroRNA-124抑制大鼠血管平滑肌细胞增殖及分子机制研究

Inhibition effect of microRNA-124 on rat vascular smooth muscle cell proliferation and its molecular mechanism

目的探讨MicroRNA-124(miR-124)对大鼠血管平滑肌细胞增殖的作用及可能的调控机制。方法选择10只11周龄雄性SPF级自发性高血压大鼠(SHR)作为实验组及同龄的10只SPF级Wistar大鼠(WKY)为对照组。采用组织贴片法原代培养SHR和WKY主动脉平滑肌细胞。通过实时荧光定量PCR(qRT-PCR)技术检测miR-124在两组主动脉平滑肌细胞间的表达差异。将miR-124模拟物或模拟物阴性对照转染SHR主动脉平滑肌细胞,分析过表达miR-124对血管平滑肌细胞增殖的影响。通过数据库及生物信息学软件预测miR-124的靶基因,并利用双荧光素酶报告基因系统进行验证。通过qRT-PCR和Western blot检测靶基因Meox2 mRNA及蛋白表达变化。采用RNA干扰技术,观察敲低Meox2表达对血管平滑肌细胞增殖的影响。结果 MiR-124在SHR主动脉平滑肌细胞中的表达为WKY主动脉平滑肌细胞的0.22倍(P<0.01)。体外过表达miR-124可明显抑制SHR主动脉平滑肌细胞的增殖。经生物信息学分析及体外双荧光素酶报告基因系统检测,证实Meox2是miR-124的靶基因。过表达miR-124后SHR主动脉平滑肌细胞中Meox2 mRNA的表达为阴性对照组的0.29倍(P<0.01);Western blot结果显示,Meox2蛋白表达水平亦明显降低。干扰Meox2的表达亦可降低SHR主动脉平滑肌细胞的增殖。结论 MiR-124在SHR中表达下调,其可能通过介导Meox2调控主动脉平滑肌细胞的增殖进而抑制高血压病变的发生过程。

Objective To investigate the inhibition effect and molecular mechanism of microRNA-124（ miR-124） on rat vascular smooth muscle cell proliferation. Methods We applied 10 male 11-weekold spontaneously hypertensive rats（ SHR） as experimental group and 10 age matched Wistar Kyoto（ WKY）rats as control group. Aortic smooth muscle cells were isolated from the medial layer of thoracic aorta of SHR and WKY rats and cultured in Dulbecco＇ s modified eagles medium（ DMEM）. Quantitative real-time PCR（ RT-PCR） was used to detect the expression of miR-124 in SHR and WKY aortic smooth muscle cells. The SHR aortic smooth muscle cells were transfected with either miR-124 mimics or NC mimics control. MTT assay was used to explore the proliferation of SHR aortic smooth muscle cells in vitro. The targeted gene of miR-124 was predicted by bioinformatics,and verified by dual luciferase reporter assay. The over-expression of miR-124 in SHR aortic smooth muscle cells was analyzed by the mRNA RT-PCR and protein expression of Meox2 by Western blot analyses. Finally,SHR aortic smooth muscle cells proliferation was also detected using MTT assay after treated with si Meox2. Results The expression of miR-124 in SHR aortic smooth muscle cells was 0. 22 times of WKY aortic smooth muscle cells（ P〈 0. 01）. Over-expression of miR-124 could significantly inhibit SHR aortic smooth muscle cells proliferation. Bioinformatics analysis and dual luciferase reporter assay demonstrated that Meox2 was a target gene of miR-124. The mRNA level of Meox2 in SHR aortic smooth muscle cells after treated with miR-124 mimics was 0. 29 times of transfected NC mimics control（ P〈 0. 01）. Western blot showed that Meox2 protein was also decreased after treated with miR-124 mimics. Knock-down Meox2 could also arrest the SHR aortic smooth muscle cells proliferation.Conclusions The expression of miR-124 is down-regulated in SHR,which may be mediated by Meox2 regulation of SHR aortic smooth muscle cells proliferation and inhibit the occurrence of hypertensive disease.