miR-133a-3p负调控NG2参与肌成纤维细胞活化的机制研究

Mechanism of miR-133a-3p Negatively Regulating NG2 in Myofibroblasts Activation

该文旨在探讨在肌成纤维细胞(myofibroblasts,MFs)活化的过程中,微小RNA(microRNA,miRNA)对神经胶质抗原2(neuron-glial antigen 2,NG2;基因名:Cspg4)表达的调控及其分子机制。小鼠骨髓间充质干细胞(bone marrow mesenchymal stromal cells,BMSCs)经miRNA mimics转染和转化生长因子β1(transforming growth factorβ1,TGFβ1)诱导后,采用qRT-PCR方法检测Cspg4以及MFs活化标志物的表达;通过双荧光素酶报告基因实验检测Cspg4与miRNA的结合情况。结果显示,MFs活化后,Cspg4的表达以时间和剂量依赖性方式上调,且与MFs的活化标志物呈正相关。敲减Cspg4后,MFs的活化标志物表达下调。在肝损伤模型中,miR-133a-3p表达下调,且与Cspg4呈显著负相关。miR-133a-3p通过与Cspg43ʹ非翻译区(3ʹuntranslated region,3ʹUTR)的特定位点结合,对Cspg4进行转录后调控。miR133a-3p可以抑制TGFβ1诱导的Cspg4和MFs活化标志物的水平升高。总之,miR-133a-3p通过负调控NG2的表达参与了MFs的活化。

The aim of this study is to explore miRNA(microRNA)regulation of NG2(neuron-glial antigen 2,gene name:Cspg4)expression and its molecular mechanism in the process of activating MFs(myofibroblasts).Mouse BMSCs(bone marrow mesenchymal stromal cells)were transfected with miRNA mimics and induced with TGFβ1(transforming growth factorβ1)to differentiate into MFs.The expression of Cspg4 and MFs activation markers were measured by qRT-PCR.The binding of Cspg4 and miRNA was detected by dual luciferase reporter gene assay.MFs activation up-regulated Cspg4 expression in a time-dependent and dose-dependent manner.The mRNA expression of Cspg4 was positively correlated with MFs activation markers.The mRNA expression of MFs activation markers were down-regulated after Cspg4 siRNA transfection.In the mouse model of liver injury,the miR-133a-3p expression was down-regulated and negatively correlated with Cspg4.miR-133a-3p binded to specific sites in the Cspg43ʹUTR(3ʹuntranslated region)for post-transcriptional regulation.TGFβ1-induced up-regulation of Cspg4 and MFs activation markers were inhibited by miR-133a-3p.In conclusion,miR-133a-3p is involved in the activation of MFs by negatively regulating the expression of NG2.