miR-27a-3p靶向表皮生长因子受体减轻脑出血引起的脑损伤

MiR-27a-3p attenuates brain injury caused by intracerebral hemorrhage through targeting epidermal growth factor receptor

目的探究miR-27a-3p减轻大鼠脑出血(intracerebral hemorrhage,ICH)引起的脑损伤的分子机制。方法将36只建模成功的SD大鼠随机分为模型组、miR-27a-3p干预组(miR-27a-3p组)和miR-27a-3p+pcDNA3.1-EGFR干预组(pcEGFR组),每组12只;另取12只健康SD大鼠作为假手术组。通过自体尾动脉血注入法构建ICH大鼠模型,假手术组健康SD大鼠只进针不注血,miR-27a-3p组ICH大鼠转染miR-27a-3p,pcEGFR组ICH大鼠转染miR-27a-3p+pcDNA3.1-EGFR。采用定量实时聚合酶链式反应(qRT-PCR)检测大鼠脑组织或血清中表皮生长因子受体(epidermal growth factor receptor,EGFR)的mRNA和miR-27a-3p的相对表达情况;采用Western blotting分析Occludin、Claudin-5和EGFR蛋白表达水平。采用免疫荧光染色对过氧化物酶(myeloperoxidase,MPO)阳性细胞进行定量分析;使用TUNEL染色分析血肿周围区域的细胞凋亡情况;通过免疫组织化学方法分析大鼠脑组织OX-42的蛋白表达情况。结果与假手术组比较,模型组大鼠血清中miR-27a-3p水平降低(P<0.001);血肿周围组织中miR-27a-3p水平降低(P<0.01)。与模型组比较,miR-27a-3p组大鼠的Occludin和Claudin-5蛋白表达水平升高(P<0.05)。与假手术组比较,模型组大鼠TUNEL阳性凋亡神经元数量、MPO阳性细胞数量和血肿周围区域OX-42阳性细胞数量均升高(P<0.01);与模型组比较,miR-27a-3p组大鼠TUNEL阳性凋亡神经元数量、MPO阳性细胞数量和血肿周围区域OX-42阳性细胞数量均降低(P<0.05)。qRT-PCR和脑含水量检测结果显示,与模型组比较,miR-27a-3p组大鼠EGFR的mRNA表达水平和脑含水量均降低(P<0.05);与miR-27a-3p组比较,pcEGFR组大鼠EGFR的mRNA表达水平和脑含水量均升高(P<0.05)。结论miR-27a-3p可显著减轻ICH诱导的脑水肿、白细胞浸润和神经元凋亡。外源性miR-27a-3p可能通过靶向紧密连接复合物和EGFR蛋白维持血脑屏障正常生理功能。

Objective To investigate the molecular mechanism of miR-27a-3p reducing brain damage caused by intracerebral hemorrhage(ICH).Methods Thirty-six SD rats with successful modeling were randomly divided into model group,miR-27a-3p intervention group(miR-27a-3p group)and miR-27a-3p+pcDNA3.1-EGFR intervention group(pcEGFR group),with 12 rats in each group.Another 12 healthy SD rats were selected as sham group.The rat ICH models were constructed by autologous tail artery blood injection.The healthy SD rats in sham group were inserted with needle but no blood injection,and the ICH rats in miR-27a-3p group were transfected with miR-27a-3p,and the ICH rats in pcEGFR group were transfected with miR-27a-3p+pcDNA3.1-EGFR.The relative expression levels of EGFR mRNA and miR-27a-3p in rat brain tissue or serum were detected by quantitative real time polymerase chain reaction(qRT-PCR).The expression levels of Occludin,Claudin-5 and EGFR protein were analyzed by Western blotting.Quantitative analysis of myeloperoxidase(MPO)positive cells was performed by immunofluorescence staining.The apoptosis in the area surrounding the hematoma was analyzed by TUNEL.The expression of OX-42 protein in rat brain tissue was analyzed by immunohistochemistry.Results Compared with sham group,the serum level of miR-27a-3p was reduced in model group(P<0.01),and the level of miR-27a-3p in the tissues around the hematoma was reduced(P<0.01).Compared with model group,the expression levels of Occludin and Claudin-5 protein in miR-27a-3p group were increased(P<0.05).Compared with sham group,the number of TUNEL-positive apoptosis neurons,the number of MPO-positive cells and the level of OX-42-positive cells in the area around the hematoma were increased in model group(P<0.01).Compared with sham group,the number of TUNEL-positive apoptosis neurons,the number of MPO-positive cells and the level of OX-42-positive cells in the area around the hematoma in miR-27a-3p group were reduced(P<0.01).Compared with model group,the mRNA expression level of EGFR and brain water content in miR-27a-3p group were reduced(P<0.05).Compared with miR-27a-3p group,the mRNA expression level of EGFR and brain water content in pcEGFR group were increased(P<0.05).Conclusion The miR-27a-3p can reduce ICH-induced cerebral edema,leukocyte infiltration and neuronal apoptosis of rat.Exogenous miR-27a-3p may maintain the normal physiological function of blood-brain barrier by targeting tight junction complex and EGFR protein.