DNM2基因内miRNA在中央核肌病病变中的作用及其机制研究

Centronuclear myopathy (CNM) is a congenital disease caused by mutation of the genes including DNM2 etc. CNM pathology is characterized by abundant myofibers with center nuclei and the molecular mechanism for the pathology has been yet determined. A featured pathology we noted is that the progressive pathology of CNM is closely associated with overexpression of DNM2 gene rather than its gene mutation. Inhibition of DNM2 intragenic miRNA expression may significantly attenuate the severity of diseased muscle. Our preliminary result shows that miR-199a-1， an intragenic miRNA of DNM2 gene is always overexpressed along with DNM2, and intervening miR-199a-1 level results in change of muscle regeneration as well as the ratio of centronuclear myofibers. We thus propose a new mechanism of CNM pathology stating that is: the pathology of CNM is primarily attributed to parallel overexpressed intragenic miR-199a-1 of DNM2 gene which causes myopathy through downstream target(s).This project plan to define this regulatory pathway and assess its contribution in the myopathy through a series of transgenic animal and cell models, and muscle physiology method as well as biochemical assays. The success of this project will leads to profoundly understanding of CNM pathogenesis and the basic processes of myogenesis.

中央核肌病（CNM）是由DNM2等基因突变引起的严重先天性肌病，表现为中央核肌纤维异常增多，目前对其分子机制知之甚少。研究发现：CNM与DNM2基因突变本身关系并不明显，而与其mRNA表达水平密切相关，降低其表达水平可明显改善病变，该现象提示CNM发病存在独特分子机制。我们前期研究发现：DNM2含有多个intragenic miRNAs,肌肉损伤时intragenic miR-199a-1常与DNM2表达同步升高，而干预miR-199a-1的水平可改变肌肉的修复进程和中央核肌纤维比例。由此我们提出:CNM可能是由DNM2的intragenic miRNA表达过高引起的,该miRNA通过调控靶基因表达延迟肌肉分化，导致中央核肌纤维比例增多。本课题将利用动物和细胞模型以及肌肉生理生化分析方法，探讨以上调控方式在CNM发病进程中的形成及其功能贡献，最终提出CNM发病新机制和治疗新思路。

MTM1基因突变导致一类先天性遗传疾病-X染色体偶联中央核肌病（X-linked Centronuclear myopathy, XLCNM），但其发病机制目前尚不清楚。我们通过构建的Mtm1敲除小鼠模型（Mtm1 -/y），发现dynamin 2基因 (Dnm2) 及其内含子中的microRNA miR-199a-1水平均显著上升。为探究miR-199a-1在XLCNM中的作用，我们又构建了miR-199a-1的敲除小鼠并与Mtm1 -/y交配产生双敲鼠。与Mtm1 -/y相比，双敲鼠寿命更长且肌肉生理功能得到明显改善。机制分析表明miR-199a-1可以通过直接抑制非肌肉肌球蛋白IIA （NM IIA）的表达，从而阻碍肌肉的分化和成熟，而其在XLCNM中的异常升高，是由于Stat3信号增强所致。我们的结果揭示了miR-199a-1在XLCNM中的重要作用并为该疾病的临床治疗提供了新的方向。

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