Muscle aging-related IncRNA LANCLI-AS1in myogenesis and muscle regeneration

肌肉衰老相关 IncRNA LANCLI-AS1 在肌生成和肌肉再生中的作用

• 批准号: 10251661
• 负责人: Myriam Gorospe
• 金额: $ 57.18万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10251661
• 关键词: Affect; Age-Years; Antisense Oligonucleotides; Area; Atrophic; Binding; Binomial Model; Biopsy; Cell Nucleus; Code; Cytoplasm; Disease Progression; Elderly; Exposure to; Gene Expression; Genome; Human; Impairment; Intervention; Limb structure; Linear Regressions; Mass Spectrum Analysis; Measures; Modeling; Molecular; Mus; Muscle; Muscle Fibers; Muscular Atrophy; Myoblasts; Play; Polyribosomes; Proteins; RNA; RNA analysis; RNA-Binding Proteins; Reporting; Role; Subcellular Spaces; Sucrose; Testing; Translating; Untranslated RNA; Up-Regulation; Work; differential expression; disability; frailty; muscle aging; muscle form; muscle regeneration; myogenesis; overexpression; oxidative damage; prevent; programs; sarcopenia; transcriptome sequencing

LANCL1-AS1 levels decline with muscle aging, increase with human myogenesis By combining the analysis of RNAs expressed in a human cultured model of myogenesis, as well as sequencing and muscle aging biopsies (GESTALT), we identified 48 differentially expressed RNAs shared with these analyses; 5 of these are lncRNAs. Many lncRNAs were shown to correlate with myogenesis and muscle regeneration, but few have known function. We found that LANCL1-AS1 was the most differentially expressed lncRNAs in this combined analysis. Moreover, LANCL1-AS1 is the second most significantly downregulated RNA by negative binomial models analysis during muscle aging. In addition, the linear regression plots showed that LANCL1-AS1 levels were significantly reduced with advancing age. RT-qPCR analysis confirmed a rise in the levels of LANCL1-AS1 during the early stages in human myogenesis, and continuous upregulation of OIP5-AS1 with advancing myogenic differentiation. Those results suggest that LANCL1-AS1 may play an important role for myogenesis, muscle regeneration and muscle aging. LANCL1-AS1 may promote myogenesis, translocates to cytoplasm during myogenesis To begin to study the role of LANCL1-AS1 in myogenesis, we silenced LANCL1-AS1 in AB678 human myoblasts. interestingly, this intervention reduced myogenesis progression in human myoblasts, as determined by measuring myotube formation. To further understand the potential mechanism by which LANCL1-AS1 affected myogenesis, we fractionated RNA through sucrose gradients. LANCL1-AS1 translocated from the nucleus to the cytoplasm during myogenic differentiation, suggesting that LANCL1-AS1 may play the different roles during myogenesis by targeting with different molecular partners on different subcellular spaces. Since the binding partner is critical for lncRNA functions, we used biotinylated antisense oligonucleotides (ASO) RNAs to pull down interacting RNA-binding proteins by mass spectrometry (ChIRP-MS). ChIRP and RT-qPCR analysis revealed that LANCL1-AS1 was very efficiently pulled down by specific LANCL1-AS1 ASOs. Recently, a few examples have been reported of lncRNAs that can be translated partially into small protein products (micropeptides). Interestingly, we also found LANCL1-AS1 is present in small polysome fractions, suggesting that LANCL1-AS1 has protein-coding potential. Notably, differentiated myoblasts have more LANCL1-AS1 is in the heavy polysome fraction, suggesting that LANCL1-AS1 may have functional mcicropeptide during myogenic differentiation. Ongoing Efforts To test the hypothesis that lncRNA LANCL1-AS1 promotes myogenesis and muscle regeneration, we are exploring these three areas: (1) whether LANCL1-AS1 can regulate human myogenesis by (1a) characterizing and annotating lncRNA LANCL1-AS1 in human myoblast and (1b) by determining whether silencing and overexpressing LANCL1-AS1 can regulate myogenesis in myoblasts. (2) the molecular mechanisms by which LANCL1-AS1 regulates gene expression during myogenesis by (2a) identifying and comparing systematically between LANCL1-AS1-driven gene expression changes during myogenesis; (2b) identifying systematically LANCL1-AS1-interacting proteins; and (2c) investigating whether lncRNA LANCL1-AS1 encodes small micropeptide. (3) the role of LANCL1-AS1 in sarcopenia and muscle regeneration in mice by (3a) comparing LANCL1-AS1 expression in muscle biopsy from sarcopenia and healthy donors and (3b) examining whether modulating LANCL1-AS1 levels affects muscle regeneration after exposure to hind limb atrophy.

LANCL1-AS1水平随着肌肉衰老而下降，随着人类肌发生的增加 通过结合对肌发生的人类培养模型以及测序和肌肉老化活检（Gestalt）中表达的RNA的分析，我们确定了与这些分析共享的48种差异表达的RNA。其中5个是lncrnas。 显示许多LNCRNA与肌发生和肌肉再生相关，但很少有已知功能。我们发现LANCL1-AS1是此组合分析中最差异表达的LNCRNA。此外，LANCL1-AS1在肌肉衰老期间通过负二项式模型分析是第二大度显着下调的RNA。此外，线性回归图表明，随着年龄的增长，LANCL1-AS1水平显着降低。 RT-QPCR分析证实，在人类肌发生的早期阶段，LANCL1-AS1的水平上升，并且随着肌源分化的前进，OIP5-AS1的持续上调。这些结果表明LANCL1-AS1可能对肌发生，肌肉再生和肌肉衰老起重要作用。 LANCL1-AS1可能促进肌发生，在肌发生过程中易位为细胞质 为了开始研究LANCL1-AS1在肌发生中的作用，我们在AB678人类成肌细胞中沉默了LANCL1-AS1。有趣的是，通过测量肌管形成确定，这种干预降低了人成肌细胞的肌发生进展。为了进一步了解LANCL1-AS1影响肌发生的潜在机制，我们通过蔗糖梯度分离RNA。在肌源性分化过程中，LANCL1-AS1从细胞核转移到细胞质，这表明LANCL1-AS1在肌发生过程中可能在不同的亚细胞空间上靶向不同的分子伴侣，从而起着不同的作用。由于结合伙伴对于lncRNA函数至关重要，因此我们使用了生物素化的反义寡核苷酸（ASO）RNA来通过质谱法（CHIRP-MS）拉下相互作用的RNA结合蛋白。 CHIRP和RT-QPCR分析表明，LANCL1-AS1被特定的LANCL1-AS1 ASOS非常有效地拉下。 最近，据报道了一些lncRNA的例子，这些例子可以部分转化为小蛋白质产物（少量肽）。有趣的是，我们还发现LANCL1-AS1存在于小多多层小部分中，这表明LANCL1-AS1具有蛋白质编码的潜力。值得注意的是，分化的肌细胞具有更多的LANCL1-AS1，位于重多层组中，这表明LANCL1-AS1在肌原性分化过程中可能具有功能性麦克辛肽。 正在进行的努力 为了检验lncRNA lancl1-as1促进肌发生和肌肉再生的假设，我们正在探索这三个领域： （1）LANCL1-AS1是否可以通过（1a）表征和注释人肌细胞中的lncRNA LANCL1-AS1和（1B）来调节人的肌发生，并通过确定沉默和过表达Lancl1-AS1是否可以调节肌细胞中的肌生成。 （2）通过（2a）通过（2a）识别和比较LANCL1-AS1驱动的基因表达在肌发生过程中，通过（2a）在肌发生过程中调节基因表达的分子机制。 （2b）识别系统的LANCL1-AS1相互作用蛋白； （2C）研究LNCRNA LANCL1-AS1是否编码小菌根。 （3）（3a）通过（3A）比较LANCL1-AS1表达在肌肉减少症和健康供体的肌肉活检中，以及（3B）检查调节LANCL1-AS1水平是否会影响肌肉再生后的肌肉再生是否会影响肌肉再生后，（3a）在肌肉活检中比较LANCL1-AS1表达在小鼠中的作用（3A）在暴露于后二小细脉后的肌肉再生是否会影响肌肉再生。