miR-145靶向抑制MMP-9表达对大鼠脑动脉血管平滑肌细胞增殖和迁移的影响

发布时间：2018-08-06 17:15

【摘要】：目的观察miR-145对大鼠脑动脉血管平滑肌细胞(VSMC)增殖和迁移的影响,并探讨其作用机制。方法利用组织贴块法分离大鼠脑基底动脉VSMC,将细胞分为3组,对照组转染空载体;miR-145组转染miR-145;miR-145+MMP-9组转染miR-145后再转染MMP-9。采用q PCR方法检测各组MMP-9 mRNA表达,Western blotting法检测MMP-9蛋白表达。向三组细胞中加入50 ng/mL TNF-α刺激细胞增殖,采用CCK8法检测细胞增殖情况,Transwell小室检测细胞迁移能力。根据Target Scan和miRanda数据库预测,野生型MMP-9的3'-UTR区域含有miR-145靶向结合片段,采用双荧光素酶报告基因检测验证miR-145和MMP-9的靶向关系。结果 miR-145组MMP-9 mRNA和蛋白表达均低于对照组(P均0.05),miR-145+MMP-9组MMP-9 mRNA和蛋白表达均高于miR-145组(P均0.01)。加入TNF-α处理后,miR-145组的细胞增殖OD值低于对照组,miR-145+MMP-9组的细胞增殖OD值高于miR-145组(P均0.01);miR-145组细胞迁移数低于对照组,miR-145+MMP-9组细胞迁移数高于miR-145组(P均0.01)。双荧光素酶报告检测结果显示,miR-145能够抑制野生型MMP-9的荧光素酶活性(P0.01),但不能抑制突变型MMP-9的荧光素酶活性。结论 miR-145能够通过下调MMP-9抑制TNF-α诱导的大鼠脑动脉VSMC增殖和迁移。
[Abstract]:Objective to observe the effect of miR-145 on the proliferation and migration of vascular smooth muscle cells (VSMC) in rat cerebral artery and to explore its mechanism. Methods VSMCs were isolated from rat basilar artery by tissue patch method. The cells were divided into three groups. The control group was transfected with miR-145 and then MMP-9 was transfected with miR-145. The expression of MMP-9 mRNA in each group was detected by Q PCR method. Western blotting was used to detect the expression of MMP-9 protein. 50 ng/mL TNF- 伪 was added to the three groups to stimulate cell proliferation. The cell proliferation was detected by CCK8 assay and the cell migration ability was detected by Transwell chamber. According to the prediction of Target Scan and miRanda database, the 3'-UTR region of wild type MMP-9 contains miR-145 targeted binding fragment. Double luciferase reporter gene detection was used to verify the targeting relationship between miR-145 and MMP-9. Results the expression of MMP-9 mRNA and protein in miR-145 group was lower than that in control group (P0. 05). The expression of MMP-9 mRNA and protein in MMP-9 group was higher than that in miR-145 group (P0. 01). After treatment with TNF- 伪, the OD value of cell proliferation in the miR-145 group was lower than that in the control group (P 0.01). The cell migration number in the miR-145 group was lower than that in the miR-145 group (P 0.01). The results of double luciferase report showed that miR-145 could inhibit the luciferase activity of wild-type MMP-9 (P0.01), but could not inhibit the luciferase activity of mutant MMP-9. Conclusion miR-145 can inhibit the proliferation and migration of rat cerebral artery VSMC induced by TNF- 伪 by down-regulating MMP-9.
【作者单位】：南华大学附属南华医院;
【基金】：湖南省医药卫生科研计划项目(C2016087)
【分类号】：R743.3

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