miRNA调控hERG基因表达的机制及其在药物获得性长QT综合征的作用研究

Cardiac arrest due to torsade de pointes (TdP) in the acquired form of drug-induced long-QT syndrome (LQTS) is potentially catastrophic event in hospital settings. Most drugs that cause this are thought to directly block a specific cardiac ion channel (hERG) that carries the rapidly activating delayed rectifier potassium current, IKr. However, the mechanism of the hERG gene upstream regulation in the drug-induced aLQTS has not been reported. Our research group is going to explore the relationship between abnormal expression of miRNA and the functionality change of hERG. 4 hERG related of miRNAs have been selected by luciferase reporter assay. Preliminary studies displayed that 4 miRNAs could decrease the expression hERG mRNA and protein by RT-PCR and western blot in HEK-hERG cells. This project will study influence of IKr characteristics by 4 miRNAs in HEK cells and the influence on hERG gene expression and IKr characteristics of miR-103, 3619 in rabbit cardiomyocytes. The expression level of differentially expressed microRNA was evaluated by qRT-PCR . Protein expression and subcellular localization were analyzed by western blotting and Confocal laser scanning microscope. The effect of miRNAs on the electrophysiological characteristics of the Ikr current was asseses by Patch-clamp technique. Since many studies demonstrated that serum levels of a tumor-expressed miRNA can distinguish patients with cancer from healthy controls, we will investigate the effect of hERG related miRNAs in aLQTS. Our aim is to establish the measurement of HERG related miRNAs in serum or plasma as an important circulating biomarkers detection of aLQTS. Furthermore we will carry out the prediction and identification of miRNA upstream transcription factor to clarify the mechanism of transcriptional regulation of miRNA expression. ?????.

aLQTS所致TdP是院内心源性猝死的重要原因之一，其中药物导致的aLQTS 最常见。药物阻滞hERG编码的Ikr是其重要发病机制。目前hERG上游调控在药物aLQTS的作用机制尚未见相关报道。研究组以miRNA和hERG关联性为切入点，已筛选出4个hERG相关miRNA。前期研究显示miRNA在 HEK细胞中可以负向调控hERG基因表达。本课题拟研究miRNA在HEK细胞中对IKr的电流特性影响，并在乳兔心肌细胞中探讨miR-103a-1、3619对ERG功能的调控作用，初步揭示miRNA对hERG功能调控的机制；明确miRNA是否参与了aLQTS的发病，分析循环miRNA作为监测aLQTS伴TdP的生物标记物的可能；开展miRNA上游转录因子的预测与鉴定，阐明miRNA表达的转录调控机制。以期探讨miRNA在药物aLQTS的作用和机制，为其早期诊断和防治提供实验依据。

长QT综合征（LQTS）的分子生物学研究是恶性心律失常发生机制的良好研究模型。研究已证实hERG基因编码的快速激活延迟整流钾电流（IKr）通道功能改变是aLQTS发生的重要机制之一。目前hERG的上游调控在aLQTS的作用机制尚未见相关报道。. 本研究前期，我们筛选了4个与hERG基因相关的miRNAs。鉴于miR-103a可在乳鼠心肌上表达且可以和鼠ERG基因结合，本课题组选择miR-103a为研究对象，继续深入研究。通过本研究，我们在多种细胞（HEK293,新生乳鼠原代心肌细胞、鼠心肌细胞系H9C2）上，通过qRT-PCR、western blot、激光共聚焦检测多层面地明确了miR-103a可以下调hERG基因蛋白质的表达，继而减少IKr电流的最大电流及尾电流的幅度。同时本研究设计了miR-103a的反义寡核苷酸序列，分析干预miRNA能否影响hERG的表达和功能。结果显示干预后，hERG蛋白的表达及电流的动力学均恢复到正常水平。这一系列结果表明miRNA可调控hERG的表达和功能，而AMOS序列可以拮抗这种作用。本研究评价以miRNA为靶点进行心律失常治疗的潜力，为LQTS 发生机制研究提供新思路，并为特异性抗心律失常药物的开发提供新途径，为临床LQTS 基因靶向治疗提供实验基础。本研究通过基因芯片筛查，发现本实验反向筛选的miR-103a和miR-3619在实验组和对照组中的表达有差异。这从另一个角度表明，miR-103a可能参与了aLQTS的发病，但是由于实验组标本偏少，未能大规模的验证。本研究还发现转录因子Sp1可以调控miR-103的转录活性，继而影响心肌细胞中miR-103相关ERG蛋白表达水平。这一结果初步揭示miRNA表达的转录调控机制，为进一步研究转录因子-miRNA-hERG基因这种调控机制提供实验基础。