The main features of Myotonic Dystrophy type 1(DM1) include myotonia, muscle weakness and atrophy. The impairment of muscle regeneration is considered as one of the main reasons of muscle wasting. However, the underlying mechanism has not been fully understood. Skeletal Satellite Cells (SSCs) are muscle precursor cells. The dysfunction of SSCs may have played an important role in the impaired muscle regeneration. In DM1, the expanded CUG RNAs (toxic RNA) sequester important splicing factors, MBNL proteins, leading to aberrant splicing of key genes. Toxic RNA can also interfere with translation through activation of CUG binding protein. MARK2, a key factor in modulating asymmetric division of SSC, is one of the MBNL target genes. We hypothesize that the sequestration of MBNL may affect the alternative splicing of MARK2 and the translation of its isoforms, which will result in the abnormal mitosis, decreased asymmetric division of SSCs and thus muscle regeneration. In this study, we will generate SSCs from normal human induced pluripotent stem (iPS) cells, DM1 iPS cells and genome-corrected DM1 iPS cells as a model to study the mechanism of SSC dysfunction in DM1. We will mainly focus on the effect of toxic RNA on MARK2 splicing, translation and distribution. The successful completion of this study will shed light on the mechanism of impaired muscle regeneration in DM1 and lay the foundation for therapeutic development using stem cells.
强直性肌营养不良1型(DM1)核心症状为肌萎缩无力和强直。肌萎缩的主要原因之一是肌再生障碍,机制尚不清。骨骼肌卫星细胞(SSC)是骨骼肌再生的来源。DM1发病机制的核心是毒性RNA,其与负责调控RNA剪接工作的MBNL结合,使mRNAs发生异常剪接,从而导致疾病发生。MARK2 是MBNL1 的主要靶基因之一,也是干细胞不对称分裂的调控因子。因此,我们推测毒性RNA通过调控SSC中MARK2的可变剪接和蛋白表达,引起SSC有丝分裂异常、极性损害和不对称分裂减少,导致SSC增殖分化异常,从而使DM1骨骼肌再生障碍。本项目拟将Pax7基因导入正常人iPS、DM1 iPS和基因修复的DM1 iPS,经分化筛选,获得正常人SSC、DM1 SSC和基因修复的DM1 SSC细胞系,观察DM1 SSC的增殖分化功能异常,并以毒性RNA、MARK2为切入点研究DM1 SSC增殖分化异常的分子机制。
骨骼肌萎缩是1型强直性肌营养不良(DM1)的临床症状之一。骨骼肌再生的衰退是肌肉萎缩的重要原因。骨骼肌卫星细胞(SSC)是骨骼肌再生的引擎。自噬增加可降低SSC的增殖能力。SSCs增殖能力的降低在DM1受损骨骼肌的早期再生中起着重要作用。发现增加受损的SSCs增殖的新方法的发现可能有助于鉴定用于治疗DM1中骨骼肌萎缩的新治疗靶标。通常认为Muscleblind-like 1(MBNL1)在DM1的发病机理中形成核RNA灶并干扰RNA剪接功能。但是,尚未报道MBNL1在DM1中SSCs增殖中的作用。在这项研究中,我们首先获得了与DM1患者来源的诱导多能干细胞(iPSC)分化的SSC,并将其用作研究DM1发病机理的细胞模型。由转录激活因子样(TAL)效应子核酸酶(TALEN)编辑的DM1 SSC显示MBNL1在细胞质中的分布增加,自噬减少,哺乳动物雷帕霉素靶蛋白(mTOR)的磷酸化水平增加以及增殖改善。此外,我们证实MBNL1过表达后,DM1 SSCs的增殖能力和磷酸化的mTOR水平得到增强,而自噬能力则下降。我们的数据还表明,mTOR的过表达抑制自噬后,DM1 SSC的增殖能力增强。最后,mTOR抑制剂的治疗逆转了由于MBNL1过表达引起的DM1 SSC的增殖能力的提高。综上所述,这些数据表明MBNL1通过抑制经由mTOR途径的自噬而逆转了DM1中SSC的增殖缺陷。
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数据更新时间:2023-05-31
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