Lnc-PPP2R1B作为增强子RNA招募剪切体复合物调控干细胞成骨分化的机制研究

干细胞研究是当今生命科学前沿和热点，而干细胞分化调控方兴未艾，尤其是LncRNA在定向分化中作用和机制尚不清楚。前期研究发现间充质干细胞定向成骨分化时Lnc-PPP2R1B显著上调；其过表达上调邻近编码基因PPP2R1B及成骨转录因子RUNX2和OSX，促进成骨分化，敲减其则抑制成骨；特异性招募剪切复合物组分hnRNPLL和SRSF1，呈相分离样定位在核斑，因此提出了Lnc-PPP2R1B通过招募HnRNPLL等调控PPP2R1B pre mRNA剪切促进干细胞成骨分化的假说。本项目拟通过基因编辑、单分子超分辨成像、Gro-seq和Micro-CT等技术查明Lnc-PPP2R1B如何通过相分离调控PPP2R1B剪切，明确其是否作为eRNA活化PPP2R1B表达，稳定下游β-catenin入核活化，促进RUNX2和OSX的转录调控成骨分化的分子机制，为治疗骨缺损以及骨退行性病变提供新的思路。

Stem cells and regenerative medicine are the frontier and hotspot in the field of life science, and the regulation of stem cell differentiation is in the ascendant. In particular, the role and mechanism of long noncoding RNA in linage commitment are unclear. It was found in our previous studies that lnc-PPP2R1B was significantly up-regulated when MSCs were induced to differentiate into osteoblasts; overexpression of lnc-PPP2R1B could up-regulate the adjacent coding genes PPP2R1B and osteogenic specific transcription factors RUNX2 and OSX, and promote osteogenic differentiation, and knockdown of the gene inhibited osteogenesis; it specifically bond with HNRNPLL and SRSF1, and it was located in the nuclear speckle in a phase separation manner. Based on these findings, a hypothesis has been proposed that lnc-PPP2R1B promotes the osteogenic differentiation of MSCs through binding HnRNPLL and regulating the alternative splicing of PPP2R1B. This project aims to find out how lnc-PPP2R1B can regulate the alternative splicing of PPP2R1B through phase separation by gene editing, single molecule super-resolution imaging, Gro-seq and micro CT at the cellular, tissue and animal levels, to determine whether it can activate the expression of PPP2R1B as an enhancer RNA, and to find out how lnc-PPP2R1B can promote the transcription of RUNX2 and OSX by regulating PPP2R1B to stabilize the entry to nucleaus of β-Catenin, thus promote the osteogenic differentiation. This study will provide new ideas for the treatment of bone defects and bone degenerative bone diseases.