Functional study of Linc-p27 and M-CSF in skeletal muscle stem and muscle regeneration

Ph.D.Adult muscle stem cells, satellite cells (SCs), as one of most important cell populations in the muscle tissue, contribute to muscle growth, maintenance and regeneration. Normally located in the niche beneath the basal lamina of myofibers in a quiescent stage, they will quickly activate to undergo proliferative expansion and differentiate to myotubes which fuse with each other to form mature myofibers upon injury or exercise. Meanwhile, a portion of activated SCs will return to quiescent stage to replenish the SCs pool.Here we identified a long non-coding RNA (lncRNA), Linc-p27 which is induced in activated satellite cell (ASC) then downregulated in differentiated satellite cell (DSC). Functionally, Linc-p27 knock out (KO) and knock in (KI) C2C12 myoblast cells showed significantly decreased proliferation due to arrest on G0/G1 phase of cell cycle. In the BaCl2 induced acute injury model, knockdown of Linc-p27 by siRNA injection led to delayed muscle regeneration in vivo. Mechanistically, we conducted RNA-pull down followed with mass spectrometry to define the possible binding proteins with Linc-p27, which led to the identification of Dhx36, a DNA/RNA helicase to specifically unwind G-quadruplex (G4). Multiple assays demonstrated the specific direct interaction between Dhx36 and Linc-p27. Consistently, SC specific deletion of Dhx36 in mouse severally inhibited muscle regeneration in vivo. Furthermore, by overlapping rG4-seq and Dhx36 Cross-Linking Immunoprecipitation followed by sequencing (CLIP-seq) datasets, we found Anp32e is bound by Dhx36 at a rG4 site of its 5’UTR region and it promotes myoblast proliferation. We further showed Linc-p27 or Dhx36 significantly enhanced the translation of Anp32e through unwinding the G4 motif. Altogether, our findings suggested that Linc-p27 binds and facilitates Dhx36 to unwind rG4 structure in 5’UTR of Anp32e to facilitate its translation and promotes myoblast proliferation.In addition to cell autonomous regulation, we studied possibly cell-cell crosstalk mediated by SCs secreted factors. By analyzing RNA-seq dataset of fresh isolated satellite cell (FISC), ASC and DSC we defined the global secretome differently released during SC activation and found macrophage colony stimulating factor (MCSF) is released by ASC. Meanwhile, by analyzing the M-CSF receptor expression with published single cell RNA-seq in peripheral-blood derived cell population and muscle derived cell population, we found that M-CSF receptor is highly expressed in macrophages. Furthermore, we demonstrated the pro-inflammatory profile of macrophage can be dampened with recombinant M-CSF. Altogether our data suggested SCs secreted M-CSF can possibly mediate the interaction between SCs and macrophages during muscle regeneration.卫星细胞（SCs）是成人的骨骼肌干细胞，它是肌肉组织中最重要的细胞类群之一，参与肌肉发育，维持和再生。通常处于静息状态并位于肌纤维基底膜下方，在损伤或运动后，它们会迅速激活并扩增分化形成肌管，这些肌管进一步融合成熟为肌纤维。同时一部分激活的卫星细胞会返回至静息状态来补充干细胞库。我们发现了一个长非编码RNA (lncRNA) Linc-p27，它在激活卫星细胞（ASC）中被诱导表达，但是在分化卫星细胞（DSC）中又降低表达。Lincp27敲除（KO）和敲入（KI）C2C12 细胞系由于G0/G1 期的停滞，导致其增殖明显降低。在BaCl2 诱导的急性损伤模型中，肌肉注射siLinc-p27 可以延迟肌肉再生。为了研究Linc-p27 的作用机制，我们做了RNA-pull down 和质谱分析，以确定与Linc-p27 可能结合的蛋白。多种实验证明Dhx36，一种可以特异性解开G4 的DNA / RNA 解旋酶，可与Linc-p27 直接相互作用。且小鼠卫星细胞特异性敲除Dhx36 也能同样地抑制肌肉再生。而且，通过重叠Dhx36 rG4-seq 和Dhx36 交联免疫沉淀数据集（CLIP-seq），我们发现了Anp32e 的5’端非翻译区的RNA G4 位置可以被Dhx36 结合，且其可以促进成肌细胞增殖。进一步研究证明Linc-p27 或Dhx36 可以促进Anp32e 翻译。总之，我们的发现表明Linc-p27 结合并促进 Dhx36 解开Anp32e 非编码区中的RNA G4 结构以促进其翻译，从而促进成肌细胞的增殖。除细胞自主调节外，我们还研究了由SCs 分泌因子所介导的细胞间的相互交流。通过分析新鲜离体卫星细胞（FISC），ASC 和DSC 的RNA-seq 数据集，我们得到了卫星细胞激活过程中差异表达的分泌组，发现了ASC 可以分泌巨噬细胞集落刺激因子（M-CSF）。同时，为了知道分泌的M-CSF 靶向何种细胞，我们用外周血衍生的细胞群和肌肉来源的细胞群中的已发表的单细胞RNA-seq 分析了M-CSF 受体的表达，发现M-CSF 受体的表达水平在巨噬细胞中最高。此外，我们证明巨噬细胞的促炎谱可以被M-CSF 抑制。总之我们的数据表明卫星细胞分泌的M-CSF 在肌肉再生过程中能介导卫星细胞和巨噬细胞间的相互交流。Xue, Guang."November 2019."Thesis Ph.D. Chinese University of Hong Kong 2020.Includes bibliographical references (leaves 140-156).Abstracts also in Chinese.Title from PDF title page (viewed on 20 May, 2021)