miRNA-491-5p调控Wnt1信号通路对萎缩性骨不连成骨分化的影响和作用机制研究

The treatment of atrophic nonunion still remains one of the challenges of traumatic orthopedics and its etilology is not clear. miRNAs play a crucial role in the modulation of osteogenic differentiation of MSC and seems a promising therapeautic target of atrophic nonunion. We have performed microarray analysis of differentially expressed miRNAs between human atrophic nonunions with standard healing fractures and find miRNA-491-5p upregulated significantly in atrophic nonunion , which was validated by qRT-PCR. Via bioinformatics prediction, Wnt1 ,the osteogenic factor ,may be the target gene of miRNA-491-5p .Our further experiment by qRT-PCR and Western blot show that miRNA-491-5p was upregulated significantly in atrophic nonunion and downregulated significantly in the process of osteogenic differentiation of BMSC ,while Wnt1 was downregulated in atrophic nonunion tissue and upregulated in the process of osteogenic differentiation of BMSC.Therefore,we assumed that miRNA-491-5p by regulating target gene Wnt1 and its downstream gene influence osteogenic differentiation of BMSC and participate in occurrence of atrophic nonunion.. The regulatory relationship between miRNA-491-5p and its putative target gene Wnt1 will be confirmed by a dual luciferase reporter assay.The expression of miRNA-491-5p , Wnt1/Rac1/MAP3K/JNK signaling factor,Wnt1/GSK3β/β-catenin/TCF/LEF signaling factor and osteogenic marker in specimens of human atrophic nonunions and standard healing fracture will be investigated by qRT-PCR and Western blot.Both loss-of-function and gain-of-function approaches will be used to analyze the influence of miRNA-491-5p on Wnt1 and its downstream genes in the process of osteogenic differentiation of BMSC. . In vivo, fracture will be established in a rat model, the role of miRNA-491-5p modulating Wnt1 signal pathway in callus formation will be investigated by X-ray, immunohistochemistry,qRT-PCR and Western blot in order to find a new effective way to prevent and treat atrophic nonunion.

萎缩性骨不连的治疗是创伤骨科难题之一，其发病机制尚不明确。miRNA是成骨分化的重要调节因子，有望成为骨不连治疗的新靶点。申请人前期基因芯片结果显示miRNA-491-5p在萎缩性骨不连组织中呈异常高表达，生物信息学预测显示Wnt1可能为miRNA-491-5p的靶基因，前期临床标本和细胞学实验已证实miRNA-491-5p与Wnt1在成骨分化过程中呈负相关。因此课题组假设miRNA-491-5p通过调控Wnt1及其下游基因导致MSC成骨分化受阻，参与萎缩性骨不连的发生。本课题拟采用荧光素酶基因报告对miRNA-491-5p与Wnt1的直接作用关系进行确认，通过体内、体外实验观察miRNA-491-5p增强和抑制对Wnt1信号通路及成骨分化的影响，探讨miRNA-491-5p调控Wnt1信号通路在萎缩性骨不连发生过程中的作用机制，寻找萎缩性骨不连治疗的新靶点。

萎缩性骨不连的治疗是创伤骨科难题之一，其发病机制尚不明确。miRNA是成骨分化的重要调节因子，有望成为骨不连治疗的新靶点。本课题应用基因芯片对萎缩性骨不连组织和正常新鲜骨折软骨痂的miRNA进行了检测分析，对高表达miRNA采用QPCR实验进行验证，发现骨不连组织中miR-381的上调最明显，进行生物信息学预测，显示miR-381可能靶向调控Wnt5A, 用荧光素酶基因报告对miR-381与Wnt5A的直接作用关系进行了确认。免疫组化实验显示骨不连组织中Wnt5A下调，同时采用QPCR、WB和ELISA方法检测miR-381增强和抑制后BMSCs的成骨基因的表达变化，发现miR-381抑制BMSCs的成骨分化，并且miR-381下调Wnt信号通路相关蛋白的表达，证实了 miR-381通过调控Wnt5A/FZD3信号通路抑制BMSCs成骨分化。本课题同时发现miR-199b-5p通过抑制GSK-3β/β-catenin信号通路调控BMSCs成骨分化miR-139b-5p通过靶向FZD4/CTNNB1蛋白调控Wnt信号通路影响成骨分化。本课题建立了动物股骨干骨折模型，采用X线和HE染色检测发现骨折端局部注射miR-491 inhibitor 可促进骨折愈合。我们认为miR-381、 miR-199b-5p和miR-139b-5p可以为萎缩性骨不连的诊断提供参考并可以作为潜在的药物治疗靶点。