陈超,张延慧,王明卉,许胜,刘翠云,李培峰,王昆.长链非编码RNA CAF调节阿霉素诱导的心肌细胞毒性机制的研究[J].转化医学杂志,2018,7(2):68-73
长链非编码RNA CAF调节阿霉素诱导的心肌细胞毒性机制的研究
Effect of the long non coding RNA CAF on Doxorubicin induced cardiotoxicity and its possible mechanisms
  
DOI:
中文关键词:  阿霉素  心脏毒性  长链非编码RNA  MIEF1
英文关键词:Doxorubicin (DOX)  Cardiotoxicity  Long non coding RNA (lncRNA)  MIEF1
基金项目:中国博士后科学基金面上项目(2016M592133);青岛市应用项目(2016069)
作者单位
陈超 青岛大学转化医学研究院 
张延慧 青岛大学转化医学研究院 
王明卉 青岛大学附属医院 
许胜 青岛大学转化医学研究院 
刘翠云 青岛大学转化医学研究院 
李培峰 青岛大学转化医学研究院 
王昆 青岛大学转化医学研究院 
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中文摘要:
      目的阿霉素(doxorubicin,DOX)作为化疗药物广泛用于治疗各种肿瘤。然而,限制其临床应用的主要因素是心脏毒性,调节DOX诱导的心脏毒性的分子组分和详细机制仍然在很大程度上不明。本研究旨在探索长链非编码RNA(long non-coding RNA,lncRNA)调节DOX诱导的心肌细胞毒性机制。方法采用高通量lncRNAs基因芯片研究与对照组样品相比较,2 μmol/L DOX诱导的细胞样品中的lncRNA的差异性表达谱。同时采用实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)方法对差异表达的lncRNA分子进行验证。6只健康纯种雄性8周龄小鼠,通过DOX注射制作心脏毒性模型小鼠(模型组);6只健康C57BL/6J小鼠饲以基础饲料为对照组;小鼠原代心肌细胞根据转染情况分为阴性对照组,心肌细胞凋亡因子(cardiac apoptosis factor,CAF)组和CAFsiRNA组。通过荧光定量PCR测定模型组与对照组小鼠MIEF1基因的表达。通过合成lncRNA的小干扰RNA作为反向对照抑制MIEF1在小鼠原代心肌细胞中的表达,应用实时荧光定量PCR验证MIEF1表达情况。结果①对lncRNA基因芯片结果进行差异性分析,实时定量PCR检测结果显示,与正常组相比较,DOX处理组中被命名为CAF的lncRNA表达显著上调(n=4;t=7.79,P<0.01)。②DOX诱导的小鼠心肌细胞毒性模型造模成功。荧光定量PCR显示,与空白对照组相比,模型组MIEF1 mRNA的表达量明显降低(n=3;t=14.978,P<0.01);不同浓度DOX处理细胞24 h 后,DOX浓度越高,MIEF1 mRNA的表达量显著降低(n=4;t2 μmol/L=33.423,P<0.01;t4 μmol/L=36.120,P<0.01;t6 μmol/L=40.205,P<0.01)。CAFsiRNA组MIEF1下调(n=4;t=12.909,P<0.01),CAF组上调(n=4;t=33.634,P<0.01)。③蛋白印迹法显示CAF组MIEF1蛋白表达水平明显低于阴性对照组(n=4;P<0.01),然而CAFsiRNA组MIEF1蛋白表达水平高于阴性对照组(n=4;t=4.892,P<0.01)。结论LncRNA CAF在心脏细胞和小鼠的心脏响应DOX的治疗下调。用DOX治疗后MIEF1的表达水平显著降低。LncRNA CAF直接靶向 51 kDa的MIEF1,并抑制其在转录水平的表达。本研究确定了一种由lncRNA CAF和MIEF1组成的新途径,其介导DOX心脏毒性,为治疗心脏保护提供了有希望的治疗策略。
英文摘要:
      ObjectiveDoxorubicin (DOX) as a chemotherapeutic drug is widely used to treat a variety of human tumors. However, a major factor limiting its clinical use is its cardiotoxicity. The molecular components and detailed mechanisms regulating DOXinduced cardiotoxicity remain largely unidentified. Thus, we explores the effect of the long non-coding RNA (lncRNA) on DOXinduced cardiotoxicity and its possible mechanisms. MethodsLncRNAs expression profles in 2 μmol/L DOXinduced cell samples compared with control group samples were studied by highthroughput microarray. The significantly differentially expressed lncRNAs were verifed by realtime quantitative PCR. Six healthy male 8week old mice,produced the model of cardiotoxicity in mice by DOX (model group); six healthy C57BL/6J mice were fed with basal diet as control group. Neonatal mouse cardiomyocytes were divided in negative control, pcDNA3-1(-)lncRNA group and siLnRNA group by their different transfection. Cardiotoxicity model and control mice were used to determine the MIEF1 expression using fluorescence quantitative PCR method. Results①According to the microarray analysis, results of RTqPCR revealed that one lncRNA, we named cardiac apoptosis factor (CAF) was upregulated significantly in the DOX group (n=4; t=7.79, P<0.01). ②DOXinduced mouse cardiotoxicity model was successfully built. The fluorescence quantitative PCR results showed that compared with the control group, the expression of MIEF1 mRNA in model group was decreased signifcantly (n=3; t=14.978, P<0.01). The higher the concentration of DOX, the expression of MIEF1 mRNA was signifcantly decreased (n=4; t2 μmol/L=33.423, P<0.01; t4 μmol/L=36.120, P<0.01; t6 μmol/L=40.205, P<0.01). MIEF1 was downregulatedin the CAFsiRNA group (n=4; t=12.909, P<0.01) and upregulated in the CAF group (n=4; t=33.634, P<0.01). ③Western Blot results showed a significant down regulation of expression of MIEF1 (n=4; P<0.01) in CAF group and a significant upregulation of expression of MIEF1 (n=4; t=4.892, P<0.01) in CAFsiRNA group compared with negative control group. ConclusionLncRNA CAF is down regulated in the cardiomyocyte and mouse heart in response to DOX treatment. The expression level of MIEF1 was significantly decreased after treatment with DOX. LncRNA CAF directly targets mitochondrial dynamics protein of 51kDa (MIEF1) and suppresses its expression at transcriptional level. Our study identified a novel pathway composed of lncRNA CAF and MIEF1 that mediates DOX cardiotoxicity. This discovery provides a promising therapeutic strategy for cardioprotection.
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