The purpose of this study is to investigate miRNAs’ effects, targeting the Wnt signaling pathway, on osteogenic differentiation to provide new targets for diabetic osteoporosis treatments. Twelve male rats were divided into a normal rat group (NOR group) and a model rat group (MOD group). Cluster analysis of differentially expressed miRNAs and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed. Primary rat bone marrow mesenchymal stem cells (BMSCs) were divided into a high-glucose group and a low-glucose group, and osteogenic differentiation was induced. Alkaline phosphatase (ALP) staining and Alizarin Red staining were used for pathological analysis of the cells. Western blot analysis was used to measure GSK-3β, β-catenin, p-β-catenin, c-Myc, and CyclinD1 expression. Immunofluorescence (IF) was used to analyze the effect of GSK-3β inhibitor (CHIR99021) on β-catenin and CyclinD1 expressions levels in BMSCs. A total of 428 differentially expressed miRNAs were found between the NOR and MOD groups. KEGG analysis showed that the target genes were mostly enriched in signaling pathways, including PI3K-Akt, focal adhesion, AGE-RAGE, HIF-1, and Wnt. qPCR verification demonstrated that miR-124-3p exhibited the greatest difference in expression level. In BMSCs, miR-124-3p overexpression could reverse the inhibited expression of BMSC osteogenic markers, including Alpl, Bglap, and Runx2, induced by high glucose. Western blot analysis revealed that the transfection of miR-124-3p mimics could further reverse the upregulated p-β-catenin and GSK-3β levels and the downregulated c-Myc and CyclinD1 levels induced by high glucose. IF results revealed that BMSCs treated CHIR99021 under high glucose showed the reduced GSK-3β and increased β-catenin and CyclinD1 expression levels. Our research highlighted miRNAs’ important roles in regulating the Wnt pathway and provided new information for the diagnosis and treatment of diabetic osteoporosis.

本研究的目的是研究 miRNA 靶向 Wnt 信号通路对成骨分化的影响，为糖尿病骨质疏松症的治疗提供新的靶点。12只雄性大鼠分为正常大鼠组（NOR组）和模型大鼠组（MOD组）。进行了差异表达的 miRNA 的聚类分析和京都基因和基因组百科全书 (KEGG) 分析。将原代大鼠骨髓间充质干细胞（BMSCs）分为高糖组和低糖组，诱导成骨分化。碱性磷酸酶(ALP)染色和茜素红染色用于细胞病理学分析。蛋白质印迹分析用于测量 GSK-3β、β-连环蛋白、p-β-连环蛋白、c-Myc 和 CyclinD1 的表达。免疫荧光 (IF) 用于分析 GSK-3β 抑制剂 (CHIR99021) 对 BMSCs 中 β-catenin 和 CyclinD1 表达水平的影响。在 NOR 和 MOD 组之间共发现了 428 个差异表达的 miRNA。KEGG分析显示靶基因主要富集于信号通路，包括PI3K-Akt、粘着斑、AGE-RAGE、HIF-1和Wnt。qPCR 验证表明 miR-124-3p 表现出最大的表达水平差异。在 BMSC 中，miR-124-3p 过表达可以逆转高糖诱导的 BMSC 成骨标志物（包括 Alpl、Bglap 和 Runx2）的抑制表达。蛋白质印迹分析显示，转染 miR-124-3p 模拟物可以进一步逆转高糖诱导的 p-β-catenin 和 GSK-3β 水平上调以及 c-Myc 和 CyclinD1 水平下调。IF 结果显示，在高糖条件下处理 CHIR99021 的 BMSC 显示 GSK-3β 减少，β-连环蛋白和 CyclinD1 表达水平增加。我们的研究突出了miRNA在调节Wnt通路中的重要作用，为糖尿病骨质疏松症的诊断和治疗提供了新的信息。