Department of Endocrinology, Shenzhen Traditional Chinese Medicine Hospital, No. 1 Futian District, Shenzhen, 518033, Guangdong, China.
In Vitro Cell Dev Biol Anim. 2020 Oct;56(9):723-734. doi: 10.1007/s11626-020-00502-0. Epub 2020 Oct 21.
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.
本研究旨在探讨 miRNA 通过靶向 Wnt 信号通路对成骨分化的影响，为糖尿病性骨质疏松症的治疗提供新的靶点。将 12 只雄性大鼠分为正常大鼠组（NOR 组）和模型大鼠组（MOD 组）。对差异表达的 miRNA 进行聚类分析和京都基因与基因组百科全书（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，并诱导其成骨分化。碱性磷酸酶（ALP）染色和茜素红染色用于细胞的病理分析。Western blot 分析用于检测 GSK-3β、β-catenin、p-β-catenin、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。NOR 和 MOD 组之间共发现 428 个差异表达的 miRNA。KEGG 分析表明，靶基因主要富集在信号通路中，包括 PI3K-Akt、黏附斑、AGE-RAGE、HIF-1 和 Wnt。qPCR 验证表明，miR-124-3p 的表达水平差异最大。在 BMSCs 中，过表达 miR-124-3p 可逆转高糖诱导的 BMSC 成骨标志物 Alp1、Bglap 和 Runx2 表达受抑制的现象。Western blot 分析显示，miR-124-3p 模拟物的转染可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 结果表明，在高糖条件下用 CHIR99021 处理的 BMSCs 显示出 GSK-3β 减少和β-catenin 和 CyclinD1 表达水平增加的现象。本研究强调了 miRNA 在调节 Wnt 通路中的重要作用，为糖尿病性骨质疏松症的诊断和治疗提供了新的信息。
