miR-134对蛛网膜下腔出血后软脑膜纤维化的作用

Effect of miR-134 on Leptomeningeal Fibrosis after Subarachnoid Hemorrhage

目的探究miR-134对蛛网膜下腔出血(SAH)后软脑膜纤维化的作用及其机制。方法大鼠(40只)按照随机数字表法随机分2组,即假手术组(SHAM组,n=20)和模型组(SAH组,n=20);2组脑组织采用芯片分析技术筛选差异表达的miRNAs,并采用RT-qPCR对其进行验证;使用miRDB软件预测miR-134的靶蛋白,采用双荧光素酶报告基因实验检测miR-134对TGF-β1的靶向作用。采用western blot和RT-qPCR检测miR-134过表达和低表达的血管内皮细胞RAOEC中TGF-β1和CollagenⅠ表达。12只SAH大鼠随机分4组,各组颅骨后分别注射空白慢病毒(空白慢病毒组,n=3)、Lv-miR-134(Lv-miR-134组,n=3)、Lv-TGF-β1(Lv-TGF-β1组,n=3)和Lv-miR-134+siRNA-TGF-β1(Lv-miR-134+siRNA-TGF-β1组,n=3),Masson染色观察各组软脑膜组织的纤维化。结果与SHAM组比较,SAH组miR-134表达降低,TGF-β1和CollagenⅠ的蛋白和mRNA表达增高(均P<0.05)。miR-134能够与TGF-β1的3′-UTR区域直接结合,并抑制其活性。血管内皮细胞中miR-134抑制TGF-β1和CollagenⅠ的蛋白和mRNA表达(P<0.05)。与空白慢病毒组相比,Lv-miR-134组、Lv-miR-134+siRNA-TGF-β1组的纤维化程度降低(P<0.05),而Lv-TGF-β1组纤维化程度升高(P<0.05)。结论miR-134通过调控TGF-β1抑制SAH后软脑膜纤维化。

Objective To investigate the effect of miR-134 on leptomeningeal fibrosis after subarachnoid hemorrhage(SAH)and its mechanism of action.Methods Forty rats were randomly divided into two groups:sham-operated group(SHAM group)and model group(SAH group),with 20 rats in each group.The differentially expressed miRNAs were screened by microarray analysis and validated using RT-qPCR.The target proteins of miR-134 were predicted using the miRDB software.The targeting effect of miR-134 on transforming growth factor-β1(TGF-β1)was analyzed by dual luciferase reporter gene experiment.The expression of TGF-β1 and collagenⅠwas detected by Western blot and RT-PCR in rat aortic endothelial cells(RAOEC)with overexpressed or underexpressed miR-134.Twelve SAH rats were randomly given lateral cerebral ventricle injection of empty lentivirus(n=3),Lv-miR-134(n=3),Lv-TGF-β1(n=3),or Lv-miR-134+siRNA-TGF-β1(n=3).Leptomeningeal fibrosis was observed by Masson staining.Results Compared with SHAM group,the expression of miR-134 decreased but the expression of TGF-β1 and collagen I increased in SAH group(P<0.05).miR-134 could be directly combined with the 3′UTR region of TGF-β1 to inhibit its activity.In RAOEC,miR-134 depressed the protein and mRNA expression of TGF-β1 and collageⅠ.Compared with empty lentivirus group,the degree of fibrosis reduced in Lv-miR-134 group and Lv-miR-134+siRNA-TGF-β1 group but enhanced in Lv-TGF-β1 group(P<0.05).Conclusion miR-134 can inhibit leptomeningeal fibrosis after SAH through regulating TGF-β1.