miR-29b通过KLF4调控血管平滑肌细胞增殖及表型蛋白表达

miR-29b Regulates Vascular Smooth Muscle Cells Proliferation and Contractile Markers by Targeting KLF4

目的探讨微小RNA-29b(microRNA-29b,miR-29b)通过靶向作用于Krüppel样因子4(Krüppel-like factor 4,KLF4)信号通路对血管平滑肌细胞增殖及表型蛋白表达的影响。方法将血管平滑肌细胞(vascular smooth muscle cells,VSMCs)分为4组:无义序列对照组(control+NC)、miR-29b过表达对照组(control+miR-29b)、无义序列模型组(ox-LDL+NC)、miR-29b过表达模型组(ox-LDL+miR-29b)。对照组细胞正常培养;模型组细胞采用80 mg/L氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导24 h建立模型。细胞计数试剂盒(cell counting kit-8,CCK-8)检测VSMCs的增殖情况;实时荧光定量PCR检测miR-29b、单核细胞化学趋化因子1(monocyte chemoattractant protein-1,MCP-1)、MCP-3、α平滑肌肌动蛋白(smooth muscleα-actin,SMα-actin)、平滑肌肌球蛋白重链(smooth muscle myosin heavy chain,SM MHC)及KLF4的mRNA表达水平;酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)试剂盒检测培养上清中MCP-1、MCP-3水平;Western blot检测蛋白表达水平;双荧光素酶报告基因实验检测miR-29b对KLF4的靶向作用。结果ox-LDL刺激VSMCs后,细胞中miR-29b表达量显著下降。与ox-LDL+NC组相比,ox-LDL+miR-29b组细胞增殖减少,炎症因子MCP-1和MCP-3的表达降低,收缩表型蛋白SMα-actin、SM MHC的表达显著升高,KLF4表达下降。双荧光素酶报告基因实验结果表明,miR-29b能与KLF4 mRNA的3′端非翻译区(3′UTR)结合。过表达KLF4可抑制miR-29b的作用。结论miR-29b可靶向作用于KLF4,调控ox-LDL诱导的VSMCs增殖、炎症因子的表达及表型蛋白表达的变化。

Objective The present study aimed to explore the effect of miR-29b on ox-LDL-stimulated vascular smooth muscle cells and the underlying mechanism.Methods VSMCs were divided into four groups:control+NC group,control+miR-29b group,ox-LDL+NC group and ox-LDL+miR-29b group.The cells were treated with 80 mg/L ox-LDL for 24 h to establish the model.A cell counting Kit-8(CCK-8)assay was performed to evaluate VSMC proliferation.The expression of miR-29b,MCP-1,MCP-3,SMα-actin,SM MHC and KLF4 was detected via quantitative reverse-transcriptase polymerase chain reaction(qRT-PCR).The expression of MCP-1 and MCP-3 in the supernatant was detected via ELISA.Protein expression was examined via Western blotting.A luciferase reporter assay was used to identify the relationship between KLF4 and miR-29b.ResultsWe found that miR-29b was downregulated significantly in ox-LDL-stimulated VSMCs.The miR-29b mimics inhibited the proliferation and expression of KLF4,MCP-1 and MCP-3 and increased the levels of SMα-actin and SM MHC in the VSMCs treated with ox-LDL.miR-29b could bind to the 3′-untranslated region(3′UTR)of KLF4 mRNA and subsequently inhibit the activity of luciferase.The overexpression of KLF4 abrogated the effects of the miR-29b mimics.Conclusion In summary,miR-29b is able to regulate the proliferation and contractile markers of VSMCs via directly regulating KLF4.