miR-363-3p调节破骨细胞在磨损颗粒诱导的骨溶解疾病的作用机制研究

Aseptic loosening is one of the most common causes of failure in artificial arthroplasty, and particle-induced osteolysis is the mainly underlying mechanism. Suppressing the differentiation and function of osteoclasts have been proven as an effective way to prevent aseptic loosening. MicroRNAs (miRs) are small non-coding RNAs that principally function in the spatiotemporal regulation of protein translation in animal cells. Recent reports about osteoclastogenesis indicated that miRs might serve as important regulators in osteoclast differentiation and function. Based on our previous research data, miR-363-3p was supposed to inhibit osteoclast differentiation and function though MITF and ITGA5, thus might serve as a potential way in the treatment of aseptic loosening. In our present research, we would firstly adopt the techniques of miRs mimics/inhibitor to testify the effects of miR-363-3p on osteoclastogenesis, bone resorption, osteoclast-related specific gene and protein expression levels; after that luciferase assay and gene over-expression techniques would be performed to explore the potential mechanisms of miR-363-3p on osteoclast; finally, we would adopt wear particles induced osteolysis skull model, synthesize and inject pre-miR-363-3p locally to evaluate the therapeutic efficacy in vivo. We aim to provide experimental basis for revealing miR-363-3p’s therapeutic effect, molecular mechanism and clinical application value.

假体周围磨损颗粒激活破骨细胞引起的骨溶解是导致人工关节无菌性松动的主要原因，靶向抑制破骨细胞预防无菌性松动成为新近研究热点。最近研究指出，miRNAs将在破骨细胞中发挥重要的调控作用。我们前期研究结果表明，miR-363-3p有望通过MITF和ITGA5负调控破骨细胞功能，提示其可用于防治磨损颗粒诱导的骨溶解。本项目拟在此基础上，采用miRNAs沉默/过表达技术，明确miR-363-3p对破骨细胞分化、功能、基因和蛋白表达的影响；其次，采用荧光素酶及转基因细胞，通过luciferase assay技术和挽救实验，验证其作用靶基因和可能的作用机制；最后，在体内建立磨损颗粒诱导的颅骨骨溶解模型，利用miRNAs沉默技术，局部注射pre-miR-363-3p，明确miR-363-3p治疗磨损颗粒诱导的骨溶解的作用。为miR-363-3p治疗该类疾病的作用、机制及应用价值提供实验依据和方法借鉴。

假体周围磨损颗粒激活破骨细胞引起的骨溶解是导致人工关节无菌性松动的主要原因。抑制假体周围破骨细胞功能，减缓骨丢失是预防磨损颗粒诱导的骨溶解、预防假体松动，延长假体使用寿命的一种有效途径。本课题以miR-363-3p 为突破口开展相关研究，抑制破骨细胞活性，减缓骨丢失，从而抑制磨损颗粒诱导的骨溶解，有望为该类疾病的治疗提供一个新视角。我们发现① miR-363-3p在破骨细胞分化过程中明显降低，过表达miR-363-3p后，破骨细胞分化及骨吸收功能受到抑制。同时TRAP、Cathepsin K、MITF和ITGA5的mRNA 表达量明显下调。而抑制miR-363-3p后，趋势与前相反。② 构建包含MITF或ITGA5质粒的RAW264.7荧光素酶细胞，我们发现miR-363-3p 对靶基因MITF和ITGA5作用有显著性差异。 ③ 构建MITF/ITGA5过稳定表达的RAW264.7细胞，转染miR-363-3p mimics或control，发现MITF/ITGA5过稳定表达可以挽救miR-363-3p导致的破骨细胞分化抑制。④ 在小鼠Ti颗粒诱导的颅骨骨溶解模型中，miR-363-3p可显著减少破骨细胞数量，抑制骨溶解发生。最终证实miR-363-3p能够有效调控破骨细胞分化和骨吸收功能；明确miR-363-3p作用的分子机制-MITF/ITGA5为其作用靶基因；明确miR-363-3p对磨损颗粒诱导骨溶解的治疗作用。此外，我们也首次发现miR-10a可靶向抑制TAK1的表达，并抑制其下游NF-κB亚基p65入核, 从而抑制破骨细胞标志性基因的表达，进而抑制破骨细胞的分化成熟及破骨细胞骨吸收功能，最终可抑制小鼠卵巢切除导致的骨质疏松。. 综上，本课题项目顺利完成，具有重要的临床转化价值和研究的可持续性。截止目前发表SCI收录论文9篇，中文会议论文1篇。