环状RNA hsa_circ_0000230 在骨不连发生发展中的作用及其机制研究

Circular RNA (circRNA) is involved in many human biological behaviors and is closely related to the occurrence and development of human diseases. Recent studies have shown that circRNA plays an important role in maintaining stem cell pluripotency and directed differentiation. However, the role of circRNA in the development of nonunion and hBMSCs osteogenic differentiation is still unknown. Based on our previous study, we found that the levels of circRNA hsa_circ_0000230 were significantly increased in nonunion tissues comparison to paired fracture union tissues. Importantly, we also demonstrated in vitro experiments that hsa_circ_0000230 could regulate the ability of osteogenic differentiation of hBMSCs. By using fluorescence in situ hybridization (FISH) technique and Luciferase report gene detection in this study, we are supposed to find that hsa_circ_0000230 can inhibit the activity of miR-200s by binding to miR-200s. Then we may investigate its specific signaling pathways and verify the target gene effect. As a result, we can get the possible mechanism of how hsa_circ_000782 affecting hBMSCs osteogenic differentiation. At the same time, we will also find the correlation between level of hsa_circ_0000230 and prognosis of nonunion. These studies will help us to understand the functional roles of hsa_circ_0000230 in the occurrence and development of nonunion，which might be a strategy for new therapeutic methods for nonunion.

环状RNA参与机体诸多生物学行为，并与人类疾病的发生发展有密切关系。新近研究表明，环状RNA在维持干细胞多能状态和定向分化中起重要作用。但关于环状RNA与骨不连发生发展的关系及在hBMSCs成骨分化中的作用尚不明确。基于前期研究，我们发现环状RNA hsa_circ_0000230在骨不连组织相对骨折正常愈合钢板周围骨痂组织表达显著增加。体外实验证实hsa_circ_0000230具有调节hBMSCs成骨分化的能力。本项目拟采用Fish技术与荧光素酶报告基因检测，明确hsa_circ_0000230与miR-200s的结合并抑制其活性，继而探明其具体信号通路，阐明hsa_circ_0000230对hBMSCs成骨分化的影响及其机制；建立hsa_circ_0000230的表达与骨不连发生发展的相关性，从一个全新角度诠释及理解骨不连发生发展的分子生物学机制，有望为骨不连的治疗提供新策略和方法。

本项目通过收集萎缩性骨不连断端间组织与骨折正常愈合后钢板内固定取出时钢板周围骨痂组织标本并分离培养骨髓间充质干细胞（hBMSCs）。随后采用circRNA芯片检测不同来源hBMSCs细胞中circRNA的差异表达，发现circRNA hsa_cirRNA_0074834的表达在骨折正常愈合组织中高于骨不连组织。将BMSCs进行成骨分化，检测BMSCs成骨分化过程中circRNA hsa_cirRNA_0074834的表达，发现circRNA hsa_cirRNA_0074834在BMSCs成骨分化过程中明显表达，并进一步通过qRCR和荧光原位杂交技术发现circRNA hsa_cirRNA_0074834均分分布于细胞核和细胞质中。构建circRNA hsa_cirRNA_0074834过表达慢病毒和敲低慢病毒，在BMSCs中过表达和敲低表达circRNA hsa_cirRNA_0074834，然后进行成骨诱导分化，circRNA hsa_cirRNA_0074834过表达能在mRNA及蛋白水平促进COL1A1，RUNX2和OCN的表达，并通过免疫荧光分析发现下调circRNA hsa_cirRNA_0074834能够抑制COL1A1，RUNX2和OCN的表达。碱性磷酸酶和茜素红染色检测hsa_cirRNA_0074834对BMSCs成骨分化的调控作用。通过RNA pull-down实验和双荧光素酶报告基因实验检测hsa_cirRNA_0074834的靶基因。最后通过异位成骨和单皮质骨缺损动物模型实验验证hsa_cirRNA_0074834在体内调控BMSCs成骨的能力。结果显示，骨不连患者BMSCs中hsa_cirRNA_0074834表达降低。hsa_cirRNA_0074834作为ceRNA，通过microRNA-942-5p调控ZEB1和VEGF的表达。hsa_cirRNA_0074834可促进BMSCs成骨分化及骨缺损修复。这些结果表明circRNA可能是治疗骨不连的关键靶点。该项目的研究成功，从一个全新角度诠释及理解骨不连发生、发展的分子生物机制，为骨不连的治疗提供新的策略和方法。