致SMN1基因Pre-mRNA异常剪接和mRNA降解的致病机制及姜黄素修正作用的研究

Proximal spinal muscular atrophy (SMA) describes a lower motor neuron disorders that can be inherited as autosomal recessive traits. SMA is caused by loss or mutation of the survival motor neuron 1 gene (SMN1). SMA represents the most common genetic cause of infant death. In our previous work, about 50% subtle mutations of SMN1 relate to the significantly reduced expression of mRNA level in SMA patients. Understanding the pathogenic mechanisms of mutations is of critical importance for therapeutic development of SMA. This present study will focus on pathogenic mechanisms of SMN1 mutations that mediate the mRNA expression deficiency, and also analyze the impact of the curcumin on the up-regulation SMN protein by alteration of pre-mRNA splicing and/or inhibition of the degradation of mRNA. ①we firstly construct the wild and mutant minigenes, by in vivo splicing assays in cultured HEK293 cells, to explore the splicing effect of mutant bases for facilitating the exons skipping. And by RNA affinity chromatography targeted to splicing regulatory factors, we further understand the disruption of the mutations on the cis-acting elements. According to the above work, we would identify the pathogenic mechanisms of the disease-associated mutations causing exon skipping. ②We establish the non-neuronal cell model from SMA patients. In the patient lines we would implement the array of translation block and analyze the basic factors of nonsense-mediated decay (NMD) pathway via siRNA gene knock-down to reveal the pathogenic mechanism of nonsense or frameshift mutations resulting in mRNA degradation. ③The curcumin, a dietary compound with potential therapeutic, is experimentally studied to correct the reduced SMN expression levels. After the cultured patients cells are treated with curcumin, the fl-SMN1 and SMN expression levels before and after the treatment would be compared. Thus, by knocking-down the splicing regulatory factors and NMD factors, we analyze the variation of fl-SMN1 and SMN expression levels. The purpose of the research is to preliminarily explore the effect of curcumine to correct aberrant splicing and inhibit NMD of disease transcripts. ④Subsequently, we carry out an array of siRNA knock-down on smn gene of VSC4.1 motor neuron of rats, to structure a rat cell model which have reduced smn expression levels (50%--60% of wild type). After the treatment with curcumin, we measure the fl-smn and smn expression levels and observe the morphological development of cultured rat neural cells. According to the results, we evaluate the correction effect of curcumin for reducing the lesion of rat neural cells. This will afford the opportunity to compare the correction changes of curcumin occurring in vitro in SMA patient cells and in the rat motor neuron cell. This project will promote the progress on pathogenic mechanism and RNA-targeted therapy of SMA.

近端脊髓性肌萎缩症（SMA）是常见的婴幼儿致死性神经肌肉病，为运动神经元存活基因1（SMN1）突变所致，我们前期工作发现约50% SMN1点突变指向mRNA表达下调。本研究，①构建微小基因在HEK293细胞进行体内剪接分析，确定突变影响外显子跳跃的剪接效应；通过RNA亲和层析分析剪接调节因子，明确突变对顺式作用元件的影响，探讨影响pre-mRNA剪接的致病机制。②建立SMA病人非神经细胞模型，应用翻译阻断和siRNA敲减无义介导降解（NMD）途径的关键因子等实验，探讨导致mRNA降解的致病机制。本研究期望推进SMA致病机制的研究。

脊髓性肌萎缩症是由于运动神经元存活基因1（SMN1）失功能所致。我们前期报道，约6.3%的SMA患儿存在SMN1碱基变异，在确定的15种变异中有10种显示患儿外周血SMN1mRNA水平显著降低。本项目我们分别靶向pre-mRNA和mRNA进行研究。我们选择位于SMN1外显子7的变异c.863G>T（p.Arg288Met）和位于内含子6毗邻3’端剪接保守序列的变异c.835-5T>G为靶点，研究SMN1外显子7跳跃的致病机制。通过构建mini基因及体外剪接分析等试验，确定上述变异造成SMN1外显子7跳跃。c.863G>T（p.Arg288Met）干扰了与剪接因子结合的ESE或形成一个新的ESE剪接元件，导致SMN1外显子7的错误剪接。在体外剪接实验中对c.835-5T>G变异分别替换了3种不同的碱基均导致了不同程度的外显子7跳跃，提示这是一个影响3’端剪接的位点。继而，以携带p.Ser8lysfs*23、p.Val19Glyfs*21和p.Leu228*变异为靶点，在病人细胞水平研究了导致SMN1 mRNA降解的致病机制。应用翻译抑制和SiRNA抑制实验，证实这些变异由于触发了NMD机制导致携带提前终止密码的mRNA在翻译起始阶段出现降解。研究提示，错义变异不仅影响肽链局部结构或蛋白多肽的稳定性，还有可能因干扰ESE/ESS影响外显子的正确剪接。而无义和移码变异更有可能导致mRNA的降解而非翻译截短蛋白。关注转录水平的变化及导致变化的原因，将为理解遗传病的致病机理打开一扇新窗。研究还提示，人们应重新审视碱基变异的致病性，并在临床应用中持有审慎的科学态度进行解读和评估。关于姜黄素干预作用的预探索研究，我们观察到姜黄素既可能通过影响SMN2的可变剪接来促进fl-SMN2表达，也有可能抑制无义介导的mRNA降解上调fl-SMN1表达。但其上调SMN的路径和作用机理有待进一步研究。