长链非编码RNA Linc-Cdkn1b对骨骼肌干细胞及肌肉再生的的调控及其分子机制研究

Skeletal muscle has a remarkable ability to regenerate after injury owing to the muscle stem cells (also called satellite cells, SCs) which normally lie quiescently beneath the muscle fiber. SC can quickly activate and proliferate following injury, then differentiate and fuse into myofiber; meanwhile a subset of SCs undergoes self-renewal and return to the quiescent state to replenish the adult stem cell pool. Each state of the myogenic lineage progression is orchestrated by complex regulatory networks of intrinsic and extrinsic factors/mechanisms. Disruption of the regulations may render their loss of ability to regenerate, contributing to muscle wasting conditions such as muscular dystrophy, cachexia, and sarcopenia that are prevalent in our society. As a recently discovered family of cellular regulators, long noncoding RNAs (>200nt non-protein coding RNAs, lncRNAs) are remarkably powerful, flexible, and pervasive in controlling major biological processes. However, despite efforts from our groups and others in the past few years, our understanding of lncRNA participation in SC activities remains largely elusive. Here we propose to study the function of a previously uncharacterized lncRNA, Linc-Cdkn1b in promoting the myogenic lineage progression. In the pilot study, loss-of-function and gain-of-function studies demonstrated a potential role of Linc-Cdkn1b in promoting SC activation and proliferation. Mechanistic elucidation revealed it binds with RNA helicase Rhau and facilitates mRNA translation by unwinding 5’UTR quadruplex (rG4) structures. These preliminary results lead us to hypothesize that Linc-Cdkn1b plays active roles in SC functionality and muscle regeneration. Three aims were proposed to (1) further elucidate how Linc-Cdkn1b regulates satellite cell behavior and muscle regeneration; (2) further dissect how Linc-Cdkn1b/Rhau regulates SC proliferation; (3) examine how Linc-Cdkn1b/Rhau regulates translation through unwinding rG4 structures. Once completed, this study will for the first time uncover Linc-Cdkn1b as a novel regulator of SC function and muscle regeneration; it will thus enhance our knowledge foundation in the intrinsic regulatory mechanisms underlying skeletal muscle regeneration for future therapeutic development of treating muscle damages and diseases.

骨骼肌干细胞（SC）是肌肉再生的主要来源。再生过程受到内源和外源因子构成的基因调控网络的精确控制,破坏该网络会导致SC失去再生能力和相关疾病的发生。近年来lncRNA在多种生理及病理过程中的重要调控作用不断被发现，但其在SC中的功能及调控机制还鲜有研究。我们发现一个在SC激活和增殖过程中高表达的Linc-Cdkn1b。实验显示它能促进SC的增殖，并且能与RNA 解旋酶Rhau结合并促进5’UTR RNA 四联体结构（rG4）的解旋和相应mRNA的翻译。因此本课题将进一步综合利用CRISPR/Cas9体内编辑，结构生物学，生化学等多种手段探索Linc-Cdkn1b的功能，分析它与Rhau协同调控SC增殖的机理，验证二者通过解旋rG4以促进转录的分子机制。课题的实施将首次揭示Linc-Cdkn1b对SC分化和肌肉再生的调控，加强我们对再生调控网络的认知，可望为相关疾病的治疗提供新的思路和靶点。

正常骨骼肌具有很强的再生能力，肌纤维在急性或者慢性情况下能迅速再生，修复损伤。成体骨骼肌干细胞（SCs）在这一过程中起着至关重要的作用。正常生理状况下SC在肌纤维基底膜下维持着静息状态，当发生肌肉损伤时， 这些静息的SCs便会被迅速激活，重新进入细胞周期变成成肌细胞并进行增殖，其中一大部分将分化融合成多核肌细胞进而成熟为新的肌纤维以修复损伤，而其中一小部分被激活的SC能够进行自我更新并回到基底膜下以维持正常状态下的干细胞数量。再生过程中每一个阶段都必须由SC细胞内及细胞外干细胞巢的分子及相关信号通路进行精密的控制以达到再生的顺利完成。SC功能失调和许多肌肉相关疾病的发生密切相关。而长链非编码RNA（lncRNA）在这一过程中的作用并不是非常清楚。在本研究中我们鉴定到Linc-Cdkn1b（现名为Lockd）是一个在骨骼肌干细胞激活过程中表达显著升高的lncRNA。体内/体外功能学研究显示这个lncRNA能够促进细胞增殖和肌肉再生。分子机制方面我们发现Linc-Cdkn1b能够直接和RNA解旋酶DHX36（又名Rhau）相互作用。作为G四联体（G-Quadruplexes，G4）的明星互作蛋白，DHX36已知能够促进mRNA 5’UTR区域G4的解旋，增强翻译的进行。通过Polysome profiling实验我们鉴定到Anp32e是Linc-Cdkn1b/DHX36的作用靶标mRNA并且Linc-Cdkn1b通过稳定DHX36和翻译起始蛋白EIF3B的结合，促进了Anp32e mRNA 5’UTR上 G4结构的解旋和蛋白的翻译。总而言之，本研究发现了Lockd/DHX36/Anp32e组成的调控路径能增强骨骼肌干细胞的增殖和肌肉再生的进行，为肌肉疾病的治疗提供了相关的理论基础和新的潜在靶点。

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