心脏钠通道无义突变的分子机制和干预策略研究

Nonsense-mediated mRNA decay (NMD) is a widespread quality control mechanism in eukaryotic cells. It can recognize and degrade aberrant transcripts harbouring a premature translational termination codon (PTC), and thereby prevent the production of C-terminally truncated proteins which might be deleterious. Nonsense-mediated mRNA decay (NMD) play a effective role as the cell monitoring mechanism to monitor cell transcripts with premature termination codon and the rapid degradation of mRNA containing a nonsense mutation. Approximately, 30% of human genetic diseases are caused by transcripts containing PTCs. These transcripts are potential targets of NMD. Uncertainty due to NMD, NMD effector will change the clinical phenotype of the disease and genetic characterization, so the NMD surveillance pathway will be highly concerned, scientists are trying to take advantage of the NMD mechanism to explore the pathologic process of the disease and design related drugs to treat these disease. On the basis of the preliminary studies, this study will perform the cardiac sodium channel as a platform to deeply explore the molecular mechanisms of nonsense mutations and intervention strategy. 1) to understand classical or non-classical NMD degradation mechanism and efficiency of nonsense mutations located on different exons, how to affect translating efficiency of nonsense mutations by intervention of the function of UPF1 or SMG1. 2) to explore the readthrough efficiency and sodium channel voltage gating of nonsense mutations with PTC124 or gentamicin or pseudouridine, as the nonsense mutations of SCN5A in different exons have great impact on the phenotype of disease. 3) to understand the functional characterization of different intervention strategies for SCN5A nonsense mutations, and to explore possible treatment strategies for different nonsense mutations.

无义介导的mRNA降解(NMD)作为一种有效的细胞监控机制，监测细胞转录产物的提前终止密码子，防止其被翻译为蛋白质。但大量的临床病例和基础研究均表明包含无义突变的mRNA也会被翻译，与NMD相互作用改变疾病的临床表型和遗传方式，所以NMD的基因监控途径备受关注，人们试图利用NMD来探索一些疾病的病理过程并设计出相应的治疗方案。本研究拟在前期研究的基础上，以心脏钠通道为平台进一步研究无义突变的分子机制和干预策略。1）了解经典或非经典NMD途径对位于不同外显子上的无义突变的降解机制及效率，干预UPF1或SMG1对NMD功能及无义突变翻译效率的影响。2）SCN5A不同部位的无义突变对疾病的表型影响很大，探导PTC124或庆大霉素或假尿苷化对SCN5A无义突变的通读效率及钠通道动力学的影响。3）了解不同干预策略对SCN5A无义突变的功能影响，针对不同的无义突变，探导可能的治疗模式。

我们的研究主要体现在二个方面：1)系统研究SCN5A无义突变的致病机制,分析不同部位的无义突变的功能及对正常转录本的影响;探导氨基糖甙类药物或PTC124或通过RNA干扰下调eRF3对SCN5A无义突变的表达效率和动力学影响。利用构建的真核表达质粒和腺病毒分别转染和感染培养的HEK293细胞和心肌细胞，用氨基糖甙类药物或PTC124或通过RNA干扰下调eRF3，处理24-72小时后，用全细胞膜片钳检测钠电流，用免疫荧光技术和蛋白印迹技术检测全长钠通道蛋白的表达情况。在细胞水平上研究SCN5A 无义突变被拯救的机制、效率和功能。发现位于不同部位或不同的碱基序列的拯救效率或拯救后的功能存在明显差异，如W1132X可以完成拯救并恢复正常的钠通道功能，而S1812X只能部分拯救却产生异常的钠通道功能。部分结果发表在Current Gene Therapy。2）了解经典或非经典NMD途径如何参与位于不同外显子上的无义突变的降解机制及效率，发现新的机制或关键分子。我们针对RBM25基因对SCN5A基因表达和剪接的影响，以及RAAS轴对RBM25表达的影响进行了一系列实验，获得了较好的预期效果。