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糖尿病小鼠心脏组织miRNA分析及靶基因检测

Detection of microRNAs and their target genes in heart of streptozotocin-induced diabetic mice

  • 摘要: 目的 检测糖尿病模型小鼠心肌组织microRNA(miRNA)表达,并对差异miRNA调控的靶基因进行初步预测及检测。方法 C57BL/6小鼠随机分为对照组和糖尿病模型组2组,以150 mg/kg链脲佐菌素腹腔一次性注射建立糖尿病心肌模型,6周后检测小鼠体重、血糖,取心脏组织,利用实时定量PCR(qRT-PCR)法检测小鼠心肌组织中miRNAs的表达,利用TargetScan、miRarnada和PicTar等数据库预测靶基因,并选择与心肌肥大或心肌纤维化相关基因进行功能分析和检测。结果 组织学观察发现,糖尿病小鼠心肌细胞明显肥大,细胞间质呈纤维化,并且心脏/体重比增高;qRT-PCR结果显示,13个miRNA差异表达,其中miR-19a、miR-19b、miR-22、miR-503、miR-467e表达上调,miR-1、miR-29a、miR-30a、miR-96、miR-101a、miR-142-3p、miR-199-5p、miR-374表达下调;心肌肥大或心肌纤维化相关基因 Bnp、Myh7 和 Ccnd1表达上调,Hadc1、Col1a1和Vcam1表达下调。结论 miRNA可能通过调控糖尿病小鼠心肌组织中靶基因参与糖尿病心肌病发病过程。

     

    Abstract: Objective To investigate the expression of microRNAs(miRNAs)in myocardial tissues of diabetic mice and to find the potential target genes controled by the miRNAs.Methods C57BL/6 mice were randomly divided into normal control and diabetes model group.The mice in diabetes model group were injected in peritoneal cavity with streptozocin(150 mg/kg).At the end of six weeks,the body weight and content of blood sugar were detected.The morphology of the heart tissue was detected using hematoxylin-eosin staining.The miRNA expressions in myocardial tissue of the mice were determined by using reverse transcription miRNA PCR array.The targets of the altered miRNAs were predicted using the database(Target Scan,miRarnada and PicTar). Then the target genes(Anp,Myh7,Hadc1,Col1a1,Ccnd1,and Vcam1)related to cardiac hypertrophy or myocardial fibrosis were analyzed with quantitative real-time PCR(qRT-PCR).Results Histological analysis of heart tissues exhibited cardiac hypertrophy and extensive interstitial fibrosis and the heart weight/body weight(HW/BW)ratio was significantly higher in diabetic group. Up-regulated miR-19a,miR-19b,miR-22,and miR-503 and down-regulated miR-1,miR-29a,miR-30a,miR-96,miR-101a,miR-142-3p,miR-199-5p,and miR-374 miRNAs were identified in diabetic mice with cardiomyopathy. Target genes were approved with bioinformatics analysis. Among the targets,the mRNA expressions of Anp,Myh7 and Ccnd1 were increased,while Hadc1,COL1A1 and Vcam1 were decreased in diabetic mice with cardiomyopathy.Conclusion The results demonstrate that miRNAs play an important role in the process of diabetic cardiomyopathy,which may mediate cardiac hypertrophy and/or myocardial fibrosis in diabetic cardiomyopathy.

     

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