circRNA_006229调控骨髓放射性损伤细胞周期阻滞的机制研究

Normal cells in bone marrow in patients with TBI pretreatment prior to transplantation inevitably will appear a certain degree of damage. G1 cell cycle arrest is one of the important adaptive response of DNA repair after radiation damage. let-7-mediated up-regulated CDKN1A expression plays an important role in cell cycle arrest. CircRNA, the newly discovered endogenous RNA with miRNA adsorption function, have caused wide attention of research and academia since 2013. Our previous study confirmed that the expression of circRNA_006229 significantly increased in marrow stroma cell in TBI radiation injury model. circRNA_006229 can be bound to let-7 thus participating in the cell cycle regulation. This project is intend to verify that circRNA_006229 can act as ceRNA absorbing let-7d, involved in the control of CDKN1A expression and participated in the inhibition of cell proliferation, and to explore the possibility of circRNA_006229 as drug targets of radiation resistance. This project is aimed at revealing the regulation of cell cycle arrest in radiation stress state, to elucidate the mechanism of radiation resistance and lay a foundation for new targets of drug researches.

骨髓移植前接受全身照射的患者骨髓中正常细胞不可避免地将出现一定程度的损伤，G1期细胞周期阻滞是辐射损伤后重要的机体适应性反应。let-7家族介导的细胞周期蛋白依赖激酶抑制剂 1A（CDKN1A）表达上调是细胞周期阻滞的重要环节。circRNA是最新发现的具有miRNA吸附功能的内源性RNA分子，自2013年来引起了学术界的研究热潮和广泛关注。课题组前期研究表明circRNA_006229在TBI小鼠模型骨髓基质细胞中表达显著上调，并具有与let-7家族配对结合位点。本项目拟通过验证circRNA_006229作为竞争性内源RNA反向吸附let-7d，参与let-7d靶向调控CDKN1A表达，抑制细胞增殖的机制，探讨circRNA_006229作为辐射抵抗药物靶点的可能性。本项目旨在揭示辐射应激状态下细胞周期阻滞的调节方式，为阐明辐射抵抗的发生机制和药物研究新靶点奠定基础。

骨髓间充质干细胞（bone marrow mesenchymal stem cell，BMMSCs）的辐射抵抗机制对于移植后的造血功能恢复及预防GVHD的发生意义重大。circRNA_014511在TBI后的小鼠BMMSCs中表达显著上调，生物信息学分析发现其具有miR-29b应答元件，miR-29b可通过调节P53的表达，因而与放射敏感性密切相关。本课题旨在证实circRNA_014511对miR-29b的内源性调节作用，并探讨对于BMMSCs放射敏感性的影响及机制。通过构建circRNA_014511过表达腺病毒载体，并将其转染BMMSCs，发现miR-29b-2-5p和P53表达水平均降低，且可被miR29b-mimics稍逆转。双荧光素酶报告实验证实了circRNA_014511可与mmu-miR-29b-2-5p结合。经流式细胞仪分析发现circRNA_014511过表达的BMMSCs细胞凋亡率显著降低，Bcl-2和Mcl-1表达水平上调。circRNA_014511转染组细胞在接受6Gy辐照后出现了G2期阻滞，且P21、GADD45A表达水平显著降低，Cylin B1显著上升。克隆形成实验发现circRNA_014511过表达细胞辐照后的生存分数显著高于对照组，放射敏感性降低。本研究证实了circRNA_014511可在体外通过结合miR-29b-2-5p抑制P53的表达，并通过影响细胞周期和细胞凋亡从而使BMMSCs的放射敏感性降低。