微小RNA-484参与肥厚型心肌病心肌纤维化的机制研究

Action mechanism of microRNA-484 involved in myocardial fibrosis in hypertrophic cardiomyopathy

目的寻找参与肥厚型心肌病心肌纤维化的关键微小RNAs(miRNAs),并进一步探讨其调控心肌纤维化的机制。方法选择2014年1月—2018年6月42例在四川大学华西医院诊断并行外科手术治疗的肥厚型心肌病患者,其中男29例、女13例,中位年龄46(15~69)岁。在患者纤维化程度不同的心肌组织,筛选异常表达的目标miRNA,并在细胞水平验证,通过调控该miRNA的表达,检测纤维化相关蛋白和靶基因的表达变化。最后通过双荧光素酶报告基因检测验证其靶向结合关系。结果miR-484在重度纤维化患者心肌组织中表达量明显升高。在细胞水平,心脏成纤维细胞在TGF-β1诱导活化后,miR-484表达量较活化前明显上调(P<0.05),且活化后的心脏成纤维细胞中纤维化相关蛋白(CollagenⅠ、α-SMA)表达量明显升高,靶基因HIPK1表达量下降(P<0.05);通过转染miR-484 antagomir抑制miR-484的表达后,纤维化相关蛋白表达量下降(P<0.05)。HIPK1的mRNA水平与转染NC antagomir组比较,无明显改变(P>0.05)。而在蛋白表达水平,转染miR-484antagomir组HIPK1表达量则明显增加(P<0.05)。双荧光素酶报告基因检测发现,转染WT-miR-484 mimics组荧光素酶活性较对照组下降(P<0.05)。结论miR-484是一种促纤维化的miRNA,可能参与了肥厚型心肌病心肌纤维化的过程,可作为调控的靶点,为心肌纤维化提供治疗策略。

Objective To search for the key microRNAs(miRNAs)involved in myocardial fibrosis in hypertrophic cardiomyopathy,and to further explore the mechanisms involved in the regulation of myocardial fibrosis.Methods Forty-two patients with hypertrophic cardiomyopathy diagnosed and treated surgically in West China Hospital of Sichuan University from January 2014 to June 2018 were selected,including 29 males and 13 females,with a median age of 46(15-69)years.In the myocardial tissue of patients with hypertrophic cardiomyopathy with different degrees of fibrosis,miRNAs with significantly different expression were screened and further verified at the cellular level.By regulating the expression of the target miRNAs,the expressions of fibrosis-related proteins and target genes were detected respectively.Finally,the target-binding relationship was verified by dual-luciferase reporter gene detection.Results miR-484 was up-regulated in severely fibrotic myocardial tissue and activated cardiac fibroblasts.After cardiac fibroblasts were activated by TGF-β1,the expression of miR-484 was significantly up-regulated,the expression of fibrosis-related proteins(CollagenⅠ,α-SMA)increased,and the expression of the target gene HIPK1 decreased(P0.05).After inhibiting the expression of miR-484 by transfection of miR-484 antagomir,the expression of fibrosis-related proteins decreased,while expression of HIPK1 was up-regulated(P0.05).The detection of dual luciferase reporter gene showed that the luciferase activity of the transfected WT-mi RNA-484 mimics group was lower than that of the control group(P0.05).Conclusion miR-484 is a pro-fibrotic miRNA that participates in the process of myocardial fibrosis by negatively regulating the expression of HIPK1.It can be used as a regulatory target to provide a therapeutic strategy for myocardial fibrosis.