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 通过结合对人类肌肉发生培养模型中表达的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 目的1：确定肌肉衰老相关的lncRNA LANCL 1-AS 1是否促进肌生成。 通过结合对在肌生成的人细胞培养模型中表达的RNA和在人肌肉老化活检（GESTALT）中表达的RNA的分析，发现LANCL 1-AS 1是最差异表达的lncRNA。 此外，通过负二项模型分析，LANCL 1-AS 1是肌肉老化中第二个最显著下调的RNA。 培养的成肌细胞的RT-qPCR分析揭示了在人肌生成的早期阶段LANCL 1-AS 1水平的升高，并且随着肌生成分化的推进LANCL 1-AS 1持续上调。 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下降。 通过RT-qPCR分析在人肌肉活检中验证了这些结果。 虽然LANCL 1-AS 1在神经细胞中表达，但其水平不随神经分化而增加，表明LANCL 1-AS 1在肌发生中特异性增加。 我们的初步结果还表明，沉默LANCL 1-AS 1减弱肌生成，通过监测肌管形成和测量肌酸激酶活性确定。 目标2：LANCL 1-AS 1与线粒体RNA结合蛋白LRPPRC相互作用，调节肌肉收缩相关基因以及线粒体编码基因。 考虑到lncRNA的功能与其相互作用伙伴密切相关，我们通过ChIRP-质谱分析鉴定了LANCL 1-AS 1相互作用蛋白。 其中，最热门的候选人是LRPPRC，一种核和线粒体RNA结合蛋白（RBP），通过驱动转录和转录后程序来调节代谢途径和线粒体稳态。 因此，我们通过ChIRP-Western和CLIP测定验证了LANCL 1-AS 1和LRPPRC之间的相互作用。 沉默LRPPRC减弱了肌生成，RNA测序分析显示，LANCL 1-AS 1的缺失降低了肌肉收缩、离子交换活性和许多ESTA编码的转录物的表达，包括MT-CO 1、MT-CO2、MT-ND 3和MT-ND 4 mRNA，这表明LANCL 1-AS 1可能与RBP LRPPRC协调发挥作用以维持线粒体稳态，肌肉收缩，从而影响肌肉质量控制和再生。 正在进行的工作和今后的计划 1. 研究LANCL 1-AS 1和LRPPRC调节线粒体功能的分子机制。 2. 目的探讨LRPPRC及其下游线粒体调控基因在健康人衰老肌肉（GESTALT）中的表达模式。 3. 探讨LANCL 1-AS 1在小鼠肌肉再生中的调控作用。

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 AIM 1: to determine if muscle aging related-lncRNA LANCL1-AS1 promotes myogenesis. By combining the analysis of RNAs expressed in a human cell cultured model of myogenesis, and in human muscle aging biopsies (GESTALT), LANCL1-AS1 was found as the most differentially expressed lncRNA. Moreover, LANCL1-AS1 was the second most significantly downregulated RNA by negative binomial model analysis in muscle aging. RT-qPCR analysis of cultured myoblasts revealed a rise in LANCL1-AS1 levels during early stages in human myogenesis, and continuous upregulation of LANCL1-AS1 with advancing myogenic differentiation. In addition, linear regression plots showed that LANCL1-AS1 levels declined significantly with advancing age. These results were validated in human muscle biopsies by RT-qPCR analysis. Although LANCL1-AS1 was expressed in nerve cells, its levels did not increase with neural differentiation, suggesting that LANCL1-AS1 increases specifically in myogenesis. Our preliminary results also showed that silencing LANCL1-AS1 attenuated myogenesis, as determined by monitoring myotube formation and by measuring creatine kinase activity. AIM 2: LANCL1-AS1 interacts with mitochondria RNA-binding protein LRPPRC and regulates muscle contraction related genes as well as mitochondria encoding genes. Given that the function of lncRNAs is closely linked to their interacting partners, we identified LANCL1-AS1-interacting protein by ChIRP-Mass Spectrometry analysis. Among them, the top candidate was LRPPRC, a nuclear and mitochondrial RNA-binding protein (RBP), which regulates metabolic pathways and mitochondria homeostasis by driving transcriptional and post-transcriptional programs. We thus validated the interaction between LANCL1-AS1 and LRPPRC by ChIRP-Western and CLIP assays. Silencing LRPPRC attenuated myogenesis, and RNA-sequencing analysis revealed that loss of LANCL1-AS1 reduced muscle contraction, ion exchange activity, and the expression of many mitochondria-encoded transcripts, including MT-CO1, MT-CO2, MT-ND3, and MT-ND4 mRNAs, suggesting that LANCL1-AS1 may functions coordinately with the RBP LRPPRC to maintain mitochondrial homeostasis, muscle contraction and thus affect muscle quality control and regeneration. ONGOING WORK AND FUTURE PLANS 1. To study the molecular mechanism whereby LANCL1-AS1 and LRPPRC regulate mitochondria functions. 2. To investigate LRPPRC and its downstream mitochondrial regulatory genes expression patterns in healthy human aging muscle (GESTALT). 3. To investigate the regulatory role of LANCL1-AS1 in mouse muscle regeneration in vivo.