摘要
目的筛选小鼠脑出血后继发性脑损伤调节因子溶血磷脂酸受体1(Lpar1)、miR-200b,观察Lpar1、miR-200b在小鼠脑出血后继发性脑损伤组织和LPS处理的小胶质细胞中的表达,并探讨miR-200b、Lpar1之间,及与促炎因子之间的关系。方法将18只C57小鼠采用简单随机方法分为假手术组(Sham组)、脑出血组(ICH组),ICH组建立小鼠脑出血后继发性脑损伤模型,取3只ICH组小鼠和3只Sham组小鼠处理侧基底节区脑组织,通过二代测序方法进行全转录组测序,利用R软件找出两组差异表达的mRNA、miRNA。利用STRING数据库对两组差异表达基因中的上调差异基因(mRNA)编码蛋白构建蛋白-蛋白相互作用网络(PPI),分析PPI中蛋白相互作用关系,并采用Cytoscape软件筛选PPI中的关键mRNA(Lpar1)。采用miRDB和Target Scan数据库对关键mRNA的上游miRNA进行预测,最后与关键mRNA呈负相关的miRNA取交集,得到靶向miRNA(miR-200b)。分别于建模后第1、3、7天,采用实时荧光定量PCR(q PCR)检测ICH组、Sham组Lpar1 mRNA和miR-200b。用LPS(100μg/m L)刺激BV-2细胞48 h后采用q PCR检测Lpar1 mRNA和miR-200b。采用miR-200b模拟物干预LPS处理的小胶质细胞,采用q PCR检测Lpar1 mRNA和促炎因子IL-6 mRNA、IL-1βmRNA、TNF-αmRNA。结果共得到ICH组小鼠及Sham组小鼠869个差异表达mRNA和106个差异表达的miRNA,与Sham组小鼠相比,ICH组小鼠中mRNA有545个下调和424个上调,miRNA有55个下调和51个上调。上调表达的Lpar1为感兴趣的关键基因,下调表达的miR-200b可能是Lpar1靶向结合的miRNA。与Sham组相比,ICH组小鼠基底节区脑组织和LPS处理的小胶质细胞中Lpar1 mRNA升高、miR-200b降低(P均<0.05),miR-200b模拟物干预的小胶质细胞中Lpar1 mRNA、IL-6 mRNA、IL-1βmRNA、TNF-αmRNA相对表达量降低(P均<0.05)。结论相比Sham组小鼠,ICH组小鼠基底节区脑组织中mRNA有545个下调和424个上调,miRNA有55个下调和51个上调。在ICH小鼠及LPS处理的小胶质细胞中,关键基因Lpar1高表达、miR-200b低表达,二者呈靶向关系。miR-200b可抑制小胶质细胞炎症反应。
Objective To screen out the regulatory factors miR-200 b and Lpar1 in mice with secondary brain injury after intracerebral hemorrhage and to observe the expression of Lpar1 and miR-200 b in the secondary brain injury tissues and microglia treated with LPS,and to explore the relationship between miR-200 b,Lpar1 and pro-inflammatory factors.Methods Eighteen C57 mice were randomly divided into the sham operation group(sham group)and cerebral hemorrhage group(ICH group)to establish the models of secondary brain injury after intracerebral hemorrhage,and the brain tissues of the ipsilateral basal ganglia of 3 mice in the Sham group and 3 mice in the ICH group were sequenced by the second generation sequencing method.Using R software to find out the differentially expressed mRNA and miRNA between the two groups.Using the STRING database to construct the interaction network of up-regulated differentially expressed gene coding proteins,the relationship between protein interactions in the network was analyzed,and the key genes in the network were screened out by Cytoscape software and the correlation network was constructed.The upstream miRNA of key genes was predicted by miRDB and Target Scan database,and finally the miRNA negatively related to the key genes were intersected.The expression levels of key genes,upstream miRNA and pro-inflammatory cytokines IL-6 mRNA,IL-1βmRNA and TNF-αmRNA were detected by q PCR.Results A total of 869 differentially expressed mRNAs and 106 differentially expressed miRNAs were obtained in the ICH group and sham group.Compared with the sham group,545 down-regulated and424 up-regulated mRNAs,55 down-regulated and 51 up-regulated miRNAs were found in the ICH group.The up-regulated Lpar1 was the key gene of interest,while the down-regulated miR-200 b might be the target binding miRNA of Lpar1.Compared with the sham group,Lpar1 mRNA increased and miR-200 b decreased in the microglia treated with LPS and in the basal ganglia region of the ICH group(both P<0.05),and the levels of IL-6 mRNA,IL-1βmRNA and TNF-αmRNA in microglia treated with miR-200 b mimics decreased(all P<0.05).Conclusions Compared with the mice in the sham group,there are 545 down-regulated and 424 up-regulated mRNAs,and 55 down-regulated and 51 up-regulated miRNAs in the ICH group.In the ICH mice and LPS-treated microglia,the key gene Lpar1 is highly expressed and miR-200 b is low expressed;miR-200 b can inhibit the inflammatory response of microglia.
作者
侯小红
周桂银
向成明
樊银春
姚声涛
HOU Xiaohong;ZHOU Guiyin;XIANG Chengming;FAN Yinchun;YAO Shengtao(The First Affiliated Hospital of Zunyi Medical University,Zunyi 563000,China)
出处
《山东医药》
CAS
2020年第27期1-5,共5页
Shandong Medical Journal
基金
国家自然科学基金资助项目(81960230)。
作者简介
第一作者:侯小红(996-),男,硕士,主要研究方向为脑血管疾病。E-mail:l549460390@qq.com;通信作者:姚声涛(l968-),男,博士,教授,主要研究方向为脑血管疾病。E-mail:YST@zmu.edu.cn。
