姜黄素调控无义突变Dmd基因表达的分子机制及其对mdx小鼠治疗研究

Duchenne muscular dystrophy is a lethal X-linked recessive disorder, caused by the mutations in the DMD gene and thus the loss of dystrophin protein. Approximate 25%-35% of patients suffering from Duchenne muscular dystrophy are caused by nonsense mutations in the DMD gene, many of whose mRNA transcripts are degraded by nonsense-mediated mRNA decay pathway. Previously, we have clarified that curcumin is able to increase a group of PTC-containing mRNA transcripts, including those of endogenous genes and NMD reporter genes. However, whether curcumin could promote expression of the PTC-containing DMD mRMA transcript in vivo is not yet known. This project is to explore the mechanism by which curcumin regulates the PTC-containing Dmd mRNA transcript in myoblast cells of the mdx mouse, by using shRNA-induced knocking down of the critical factors in the NMD pathway. Moreover, the effect of curcumin in combination with PTC read-through compounds on the dystrophic phenotype of the mdx mice will also be assessed by analyses of gene expression, immunohistochemistry, immunofluorescence histochemistry，ELISA and muscle function. This study will not only answer if curcumin is capable of being a promissing candidate for the therapy of monogenetic diseases caused by nonsense mutaions resulting from the substitution of single base pair, but also promote the translation of curcumin from bench to clinical application.

杜氏肌营养不良症（DMD）是抗肌萎缩蛋白缺失导致的一种致死性X-连锁隐性遗传病，目前此病尚无有效临床治疗方法。约25%-35% DMD患者是由DMD基因无义突变所致，其中多数可使其mRNA经无义介导的mRNA降解（NMD）途径降解。我们前期发现，姜黄素可通过抑制NMD途径进而增加一部分包含提前终止密码（PTC+）mRNA的表达。然而，姜黄素是否可以通过抑制NMD而增加PTC+-DMD mRNA的表达仍是未知的。本研究基于mdx小鼠，在细胞水平利用shRNA敲降NMD途径关键因子，明确姜黄素调控PTC+-Dmd mRNA表达的分子机制；再通过基因表达、免疫荧光、ELISA和肌肉功能分析，评价姜黄素及PTC通读药物联合应用对mdx小鼠肌营养不良表型的治疗效果。本研究不仅能够回答姜黄素是否能作为一种治疗无义突变所致遗传病的有效药物，而且也能为将来姜黄素对此类遗传病治疗的临床转化提供理论依据。

杜氏肌营养不良症（DMD）一种致死性X-连锁隐性遗传病，约25%-35%的患者DMD基因中存在无义突变，导致肌肉组织中抗肌萎缩蛋白缺失，目前尚无有效临床治疗方法。我们前期在对其他疾病的研究中发现姜黄素可通过抑制无义介导的mRNA降解（NMD）途径进而增加患者皮肤呈纤维细胞中包含提前终止密码（PTC+）的突变mRNA表达。然而，姜黄素是否可以通过抑制NMD通路而增加DMD患者PTC+-DMD mRNA的表达仍是未知的。我们用携带无义突变的雄性mdx小鼠及野生型雄性小鼠，分别构建原代myoblast细胞，通过shRNA敲降NMD途径关键因子UPF1的表达，发现PTC+-Dmd转录本表达显著增加，提示其可通过NMD通路被降解；姜黄素可以增加mdx小鼠肌肉组织和体外培养的myoblast细胞中PTC+-Dmd转录本的水平，同时下调NMD通过多个UPF关键因子的表达；姜黄素与PTC通读药物联合应用可上调Dp116 isoform的蛋白水平；在整体水平，姜黄素给药后可显著降低mdx小鼠腓肠肌中央核肌纤维的比例并延长其前肢悬吊时间。综合以上研究结果，姜黄素能够增加mdx小鼠体内无义突变转录本的表达，并显著改善其肌肉功能，提示姜黄素有治疗无义突变所致遗传病的潜能。