微小RNA-30a通过诱导血管内皮间质化促进下肢动脉硬化闭塞症血管内膜增生

MicroRNA-30a promotes intimal hyperplasia in arteriosclerosis obliterans by inducing endothelial-to-mesenchymal transition

目的探讨微小RNA(miRNA,miR)-30a通过诱导血管内皮细胞间质化(EndMT),促进血管内膜增生,影响下肢动脉硬化闭塞症(ASO)进程。方法苏木精-伊红染色法(HE)检测ASO病变动脉形态、内膜中膜厚度;免疫荧光检测EndMT标志物表达。肿瘤坏死因子-α(TNF-α)处理人脐静脉内皮细胞(HUVECs),构建EndMT细胞模型。将体外培养的HUVECs分为转染miR阴性对照(miR-NC组)和转染miR模拟物(miR-30a组)。实时定量反转录聚合酶链反应(RT-qPCR)、蛋白质印迹法(Western blot)检测EndMT标志物表达。细胞增殖实验检测HUVECs(EndMT)增殖能力、小室细胞迁移实验、划痕实验检测其迁移能力。采用GraphPad Prism 7进行统计学分析,t检验分析实验结果。结果ASO动脉内膜增生且呈间质化改变[内膜厚度,对照组(178.40±29.61)μm,ASO(422.10±39.47)μm,t=4.940,P<0.01,n=6;中膜厚度,对照组(315.80±48.46)μm,ASO(656.80±43.91)μm,t=5.200,P<0.01,n=6],差异有统计学意义,miR-30a在HUVECs(EndMT)和ASO内膜中高表达,miR-30a表达上调能够抑制HUVECs的内皮标志物表达,同时促进其间质标志物表达[CD31,对照:3.535±0.116,miR-30a:1.360±0.185,t=9.966,P<0.01,n=3;人血管内皮钙黏蛋白(VE-Cadherin),对照:2.453±0.075,miR-30a:0.793±0.106,t=12.830,P<0.01,n=3;α-平滑肌肌动蛋白(α-SMA),对照:0.792±0.121,miR-30a:6.296±0.380,t=13.820,P<0.01,n=3;Ⅰ型胶原(COL1),对照:0.2413±0.063,miR-30a:2.859±0.142,t=16.840,P<0.01,n=3],差异有统计学意义,并促进HUVECs(EndMT)的增殖迁移能力[细胞迁移率,对照:(50.160±1.210)%,miR-30a:(68.560±1.918)%,t=8.115,P<0.01,n=6],差异有统计学意义。结论miR-30a在ASO增生内膜中表达上调,miR-30a能够诱导内皮细胞间质化,增强其增殖迁移能力,导致ASO内膜增生、促进ASO病情进展。

Objective Vascular endothelial-to-mesenchymal transition(EndMT)may play an important role in atherogenesis.The detail mechanism of EndMT in the process of arteriosclerosis obliterans(ASO)is largely unknown.In this study,we aim to characterize the role of miR-30a-induced EndMT during atherogenesis.Methods Hematoxylin-eosin(HE)staining was used to detect the tissue morphology and intima-media thickness of ASO diseased arteries.Immunofluorescence was used to detect the expression of endothelial biomarker CD31,mesenchymal biomarkerα-smooth muscle actin(α-SMA)and EndMT associated transcription factor snail.Tumor necrosis factor-α(TNF-α)was used to induce EndMT in human umbilical vein endothelial cells(HUVECs).The miR-NC and miR-30a mimics were transfected into HUVECs as control and miR-30a groups,respectively.Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to detect the expression of miR-30a and mesenchymal biomarkers.RNA in situ hybridization was used to detect the expression of miR-30a in artery tissues.RT-qPCR and Western blotting were used to detect the change of epithelial and mesenchymal biomarkers induced by miR-30a overstressing.Cell proliferation and migration changes in miR-30a overexpressed HUVECs were measured by Cell Counting Kit-8(CCK-8),wound healing and Transwell assays.T test was applied for statistical analysis using GraphPad Prism 7.Results Down-regulation of CD31 and up-regulation ofα-SMA accompanied with increased miR-30a were observed in ASO arterial intima and TNF-α-treated HUVECs[intimal thickness,CRTL=(178.40±29.61)μm,ASO=(422.10±39.47)μm,t=4.940,P<0.01,n=6;medial thickness,CRTL=(315.80±48.46)μm,ASO=(656.80±43.91)μm,t=5.200,P<0.01,n=6].Overexpression of miR-30a induced the expression of mesenchymal biomarkers(CD31,CRTL=3.535±0.116,miR-30a=1.360±0.1846,t=9.966,P<0.01,n=3;VE-Cadherin,CRTL=2.453±0.075,miR-30a=0.793±0.106,t=12.830,P<0.01,n=3;α-SMA,CRTL=0.792±0.121,miR-30a=6.296±0.380,t=13.820,P<0.01,n=3;COL1,CRTL=0.241±0.063,miR-30a=2.859±0.142,t=16.840,P<0.01,n=3),and promoted proliferation and migration of HUVECs[cell migration rate,CTRL=(50.160±1.210)%,miR-30a=(68.560±1.918)%,t=8.115,P<0.01,n=6].Conclusion MiR-30a is significantly up-regulated through the intimal hyperplasia process of ASO.The miR-30a can promote EndMT of HUVECs and enhance its proliferation and migration,which in turn leads to the ASO intimal hyperplasia and contributes to the progression of ASO.