miR-19a对腹主动脉瘤血管平滑肌细胞增殖迁移及炎症浸润的影响

Effect of miR-19a on proliferation and migration of vascular smooth muscle cells and inflammatory infiltration in abdominal aortic aneurysm

目的:探讨miR-19a对腹主动脉瘤(AAA)血管平滑肌细胞增殖迁移及炎症浸润的影响及其机制。方法:收集AAA发生后未行任何治疗的15例患者血清,15例同期健康体检者血清,以及15例手术切除AAA患者的腹主动脉瘤组织和15例正常腹主动脉组织。RT-qPCR检测miR-19a的表达水平;CCK-8检测血管平滑肌细胞(VSMCs)增殖活力;Transwell检测VSMCs迁移能力;流式细胞术检测VSMCs凋亡水平;ELISA法检测炎症因子的表达水平;Western blot检测蛋白的表达水平;双荧光素酶报告基因验证miR-19a和CDKN2B的靶向关系。结果:与对照组比,miR-19a在AAA患者组织和血清中均明显高表达。敲降miR-19a可明显抑制VSMCs增殖、迁移并诱导细胞凋亡,下调THP-1细胞炎症因子和基质金属蛋白酶(MMPs)的表达水平。双荧光素酶报告基因结果显示,miR-19a通过结合CDKN2B基因的3’非编码区(3’ UTR),进而抑制其表达水平。过表达CDKN2B可明显缓解miR-19a对VSMCs增殖和转移的抑制作用,以及THP-1细胞的炎症反应。结论:敲降mi R-19a可通过抑制VSMCs增殖和迁移,以及下调炎性细胞浸润缓解AAA的发展进程,其机制是通过靶向上调CDKN2B的表达水平。

Objective:To investigate the effect of miR-19a on proliferation and migration of vascular smooth muscle cells and inflammatory infiltration through targeting CDKN2B in abdominal aortic aneurysm(AAA)and its underlying mechanism.Methods:Serum samples were collected from 15 patients without any treatment after primary AAA,15 healthy persons during the same period,and samples of AAA tissues and without AAA tissues were collected from 15 patients.RT-qPCR was used to detect the expression of miR-19a.CCK-8,Transwell,and flow cytometry were respectively performed to examine the proliferation,migration and apoptosis of vascular smooth muscle cells.Inflammatory cytokines secretion of THP-1 cells were evaluated using enzyme-linked immunosorbent assay(ELISA).The expression of protein was measured by western blot.Dual-luciferase reporter gene assay was applied to verify the interaction relationship between miR-19a and CDKN2B.Results:Compared with the control group,miR-19a was significantly overexpressed in the serum samples and tissues of AAA patients.Knockdown of miR-19a markedly suppressed cell proliferation,migration and induced apoptosis of vascular smooth muscle cells.Silencing of miR-19a significantly decreased the levels of inflammatory cytokines and matrix metalloproteinase.Dual-luciferase reporter gene assay results showed that miR-19a decreased the expression of CDKN2B by binding to its 3’-untranslated region(UTR).Upregulation of CDKN2B attenuated the inhibitory effect of miR-19a-elevated on the proliferation and migration of vascular smooth muscle cells and inflammatory response of THP-1 cells.Conclusion:Knockdown of miR-19a could alleviate the development of AAA by inhibiting the proliferation and migration of smooth muscle cells and down regulating inflammatory cell infiltration,which its mechanism is to target up-regulation of CDKN2B expression.