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

LANCL1-AS1水平随肌肉衰老而下降，随人类肌肉发生而升高 通过结合对人类培养的肌肉发生模型中表达的RNA的分析，以及测序和肌肉老化活检(格式塔)，我们发现了48个与这些分析相同的差异表达的RNA；其中5个是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。我们发现在这一联合分析中，LANCL1-AS1是表达差异最大的lncRNAs。此外，通过负二项模型分析，LANCL1-AS1是肌肉衰老过程中第二显著下调的RNA。此外，直线回归图显示，LANCL1-AS1水平随着年龄的增长而显著降低。RT-qPCR分析证实，LANCL1-AS1在人类肌肉发生的早期阶段水平升高，并且随着成肌细胞分化的推进，OIP5-AS1的表达持续上调。这些结果提示LANCL1-AS1可能在肌肉发生、肌肉再生和肌肉衰老中发挥重要作用。 LANCL1-AS1可能促进肌发生，在肌发生过程中移位到细胞质 为了开始研究LANCL1-AS1在肌肉发生中的作用，我们沉默了AB678人成肌细胞中的LANCL1-AS1。有趣的是，通过测量肌管的形成来确定，这种干预减少了人类成肌细胞的肌肉发生进展。为了进一步了解LANCL1-AS1影响肌肉发生的潜在机制，我们通过蔗糖梯度对RNA进行了分级。在成肌分化过程中，LANCL1-AS1从胞核移位到胞浆，提示LANCL1-AS1可能通过靶向不同亚细胞空间的不同分子伴侣，在肌细胞发生过程中发挥不同的作用。由于结合伙伴对lncRNA的功能至关重要，我们使用生物素化的反义寡核苷酸(ASO)RNA通过质谱仪(Chirp-MS)下调相互作用的RNA结合蛋白(Chirp-MS)。Chirp和RT-qPCR分析表明，LANCL1-AS1能被特异的LANCL1-AS1 ASO有效地下调。 最近，有几个例子报道了lncRNAs可以部分翻译成小蛋白产物(微肽)。有趣的是，我们还发现LANCL1-AS1存在于小的多聚体组分中，这表明LANCL1-AS1具有蛋白质编码潜力。值得注意的是，分化的成肌细胞有更多的LANCL1-AS1在重多聚体部分，提示LANCL1-AS1在成肌分化过程中可能具有功能性的微肽。 持续的努力 为了验证lncRNA LANCL1-AS1促进肌肉发生和肌肉再生的假设，我们探索了以下三个领域： (1)LANCL1-AS1能否通过(A)鉴定和注释成肌细胞中的LncRNA LANCL1-AS1以及(1b)通过确定沉默和过表达LANCL1-AS1是否能够调节成肌细胞中的肌发生来调节人的肌肉发生。 (2)LANCL1-AS1在肌肉发生过程中调控基因表达的分子机制：(2a)系统地鉴定和比较LANCL1-AS1在肌肉发生过程中驱动基因表达的变化；(2b)系统地鉴定LANCL1-AS1相互作用的蛋白质；(2c)研究lncRNA LANCL1-AS1是否编码小微肽。 (3)通过(3a)比较LANCL1-AS1在骨骼肌减少症和健康供体肌肉活检中的表达，以及(3b)检测调节LANCL1-AS1水平是否影响暴露于后肢萎缩后的肌肉再生，以探讨LANCL1-AS1在小鼠骨骼肌减少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.