模拟失重下lncRNA-5233参与骨髓间充质干细胞成骨向分化的功能与机制研究

It is important for understanding the activation and development of bone loss induced by microgravity to elucidate osteogenic differentiation of osteogenic cell lines under simulated microgravity. Our previous studies show that numerous lncRNAs in osteo-differentiation process under simulated microgravity were changed greatly. However, there are no reports of lncRNAs expression pattern in microgravity induced osteogenic development obstacle and whether microgravity mediated osteoblast differentiation relies on the modulation of lncRNAs expression remains unclearly..Pre-research showed that experssion of lncRNA-5233 in bone mesenchymal stem cells was decreased when exposed to simulated microgravity while increased when induced to ostegenic differentiation. And the nearby gene of lncRNA-5233 on chromosome, CBP, was also decreased under simulated microgravity. Bioinformatic anlysis revealed several targeted binding site of miR-132-3p on lncRNA-5233 transcript. Coincidentally, CBP was the cofactor of EP300 protein which was confirm as one of the target genes of miR-132-3p and could acetylate the osteoblast differentiation key factor Runx2 protein. .To clarify the pre-research results, we are going to make clear of the expression and function of lncRNA-5233 on BMSCs osteogenic differentiation when exposed to the simulated microgravity and validate the possible mechanical based regulatory network in which lncRNA-5233 affect the expression of CBP/EP300 protein complex, kinds of acetylase that could promote acetylation of Runx2, through cis-regulatory pattern with its nearby CBP mRNAs and ceRNA regulatory mechanism with miR-132-3p. This study is aimed to provide comprehensive understanding of lncRNA function in mechanical signal mediated osteoblast differentiation and provide a new perspective toward the effects of microgravity on osteoblast function.

阐明模拟失重条件下成骨分化调控机制对于理解失重性骨质丢失的发生发展具有重要意义。我们发现，模拟失重下成骨细胞中lncRNA表达发生显著改变，但其作用尚不清楚。预实验显示，骨髓间充质干细胞中lncRNA-5233在模拟失重下表达降低，同时，染色体上lncRNA-5233的邻近基因CBP表达也显著下调；生物信息学分析显示lncRNA-5233上具有成骨分化负调控因子miR-132-3p的结合靶点，CBP又是miR-132-3p靶基因EP300的协同结合蛋白，而EP300是成骨分化关键因子Runx2的乙酰化酶。基于上述发现，本课题拟明确：模拟失重下lncRNA-5233是否能通过顺式调控邻近基因CBP表达和竞争性吸附miR-132-3p而影响EP300表达等双重作用调控CBP/EP300乙酰蛋白酶复合物活性，进而通过影响Runx2乙酰化而参与成骨分化调控。

骨骼的组织结构具有功能适应性，其生长、发育和重建都离不开力学信号的刺激，长期卧床病人或航天失重环境中的航天员会因运动或重力刺激的减弱而出现持续发展的骨代谢失衡以及骨质丢失，严重影响人体健康和骨骼功能，尤其是在我国载人航天事业飞速发展的今天，失重性骨质丢失已成为航天失重环境所导致的机体生理变化中仍未克服的主要难题。长期以来，人们对失重环境下骨骼相关细胞的功能基因和蛋白表达变化及其参与的信号通路进行了细致的研究，但仍未能阐明失重性骨质丢失的发生原因和发展机制，针对性开发的防护手段还未能有效解决失重性骨质丢失的防护问题。近年来，miRNA和long non-coding RNA（lncRNA）等非编码RNA在细胞生物发育和功能调控中的作用日趋受到重视。基于前期研究基础，本课题探索研究了模拟失重环境下lncRNA-5233参与骨髓间充质干细胞成骨向分化的功能与机制。经研究发现，lncRNA-5233是一种长链非编码RNA，其可以在mBMSCs成骨向分化过程中发挥正性调控作用，模拟失重环境下lncRNA-5233表达水平的降低可以导致分化的mBMSCs内miRNA-132-3p活性水平升高，这会抑制乙酰化转移酶EP300的蛋白翻译过程，使得HOXA10在结合到Runx2的启动子区后可招募的乙酰化转移酶复合体EP300/CBP减少，影响了成骨分化调控关键因子Runx2的转录激活，从而导致mBMSCs成骨分化障碍。这些结果揭示了模拟失重环境下lncRNA的重要作用及lncRNA-miRNA-mRNA的交互作用关系，对于进一步理解失重性骨质丢失发生发展过程中细胞分子调控机制以及寻找新的防护研究靶点具有重要意义。