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

LANCL 1-AS 1水平随着肌肉老化而下降，随着人类肌生成而增加 通过结合对在人类培养的肌生成模型中表达的RNA的分析，以及测序和肌肉老化活检（GESTALT），我们鉴定了与这些分析共有的48种差异表达的RNA;其中5种是lncRNA。 许多lncRNA被证明与肌肉发生和肌肉再生相关，但很少有已知的功能。我们发现LANCL 1-AS 1是在该组合分析中差异表达最大的lncRNA。此外，通过负二项模型分析，LANCL 1-AS 1是肌肉老化过程中第二个最显著下调的RNA。此外，线性回归图显示LANCL 1-AS 1水平随着年龄的增长而显著降低。RT-qPCR分析证实了在人肌生成的早期阶段LANCL 1-AS 1水平的升高，以及随着肌生成分化的推进OIP 5-AS 1的持续上调。这些结果表明，LANCL 1-AS 1可能在肌肉发生、肌肉再生和肌肉老化中发挥重要作用。 LANCL 1-AS 1可能促进肌细胞的发生，并在肌细胞发生过程中转位至细胞质 为了开始研究LANCL 1-AS 1在肌发生中的作用，我们沉默了AB 678人成肌细胞中的LANCL 1-AS 1。有趣的是，如通过测量肌管形成所确定的，这种干预减少了人成肌细胞中的肌发生进展。为了进一步了解LANCL 1-AS 1影响肌生成的潜在机制，我们通过蔗糖梯度分离RNA。LANCL 1-AS 1在成肌分化过程中从细胞核转位到细胞质，提示LANCL 1-AS 1可能通过与不同的分子伴侣作用于不同的亚细胞空间而在成肌过程中发挥不同的作用。由于结合伴侣对lncRNA功能至关重要，我们使用生物素化反义寡核苷酸（阿索）RNA通过质谱（ChIRP-MS）下拉相互作用的RNA结合蛋白。ChIRP和RT-qPCR分析显示，LANCL 1-AS 1被特异性LANCL 1-AS 1 ASO非常有效地拉低。 最近，已经报道了一些lncRNA可以部分翻译成小蛋白产物（微肽）的例子。有趣的是，我们还发现LANCL 1-AS 1存在于小的多核糖体组分中，这表明LANCL 1-AS 1具有蛋白质编码潜力。值得注意的是，分化的成肌细胞具有更多的LANCL 1-AS 1在重多核糖体部分中，表明LANCL 1-AS 1可能在成肌分化期间具有功能性微肽。 持续努力 为了验证lncRNA LANCL 1-AS 1促进肌肉发生和肌肉再生的假设，我们正在探索这三个领域： (1)LANCL 1-AS 1是否可以调节人肌生成，通过（1a）表征和注释人成肌细胞中的lncRNA LANCL 1-AS 1和（1b）确定沉默和过表达LANCL 1-AS 1是否可以调节成肌细胞中的肌生成。 (2)通过（2a）系统地鉴定和比较肌肉发生过程中LANCL 1-AS 1驱动的基因表达变化;（2b）系统地鉴定LANCL 1-AS 1相互作用蛋白;和（2c）研究lncRNA LANCL 1-AS 1是否编码小微肽，LANCL 1-AS 1在肌肉发生过程中调节基因表达的分子机制。 (3)通过（3a）比较来自少肌症和健康供体的肌肉活组织检查中的LANCL 1-AS 1表达和（3b）检查调节LANCL 1-AS 1水平是否影响暴露于后肢萎缩后的肌肉再生，来确定LANCL 1-AS 1在小鼠中的少肌症和肌肉再生中的作用。

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.