Background Diabetes reduces the osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMMSCs), and miRNAs are essential in this process, during which miR-145-5p plays a vital role in osteogenic and chondrogenic differentiation of cells. However, the effect of miR-145-5p on the osteogenic differentiation of diabetic BMMSCs remains unclear.
Objective To compare the osteogenic differentiation ability of BMMSCs derived from normal and type 2 diabetic rats respectively, and preliminarily explore the differences in the expression of miR-145-5p, its potential target gene SEMA3A and the key protein β-catenin of Wnt pathway in osteogenic differentiation.
Methods Twelve GK rats were fed on high-fat and high-sugar diet to establish type 2 diabetes rat model as the experimental group. Twelve Wistar rats were fed on standard diet and served as the control group. Rat femur was isolated under aseptic condition. BMMSCs derived from normal and type 2 diabetic rats (WT-BMMSCs and GK-BMMSCs) were cultured by whole bone marrow culture method. CCK-8 was used to detect the cell proliferation. Colony formation ability was detected by the crystal violet stain assay. Alkaline phosphatase activity was detected by alkaline phosphatase staining and semi-quantitative analysis. Alizarin red staining was used to detect the formation of the mineralized matrix. The expression levels of bone-related markers, miRNA-145-5p, SEMA3A and β-catenin were detected by qRT-PCR and Western blot.
Results When the stem cells were cultured for 4-9 days, the proliferation of GK-BMMSCs was significantly lower than that of WT-BMMSCs (P<0.01). On day 10, colony formation rate was significantly lower than that of WT-BMMSCs (P<0.01). On day 7 of osteogenic induction, alkaline phosphatase activity of GK-BMMSCs (P<0.001), osteogenesis-related markers alkaline phosphatase (ALP) gene (P<0.01) and protein (P<0.001) expressions, osteocalcin (OCN) gene (P<0.001) and protein (P<0.001) expressions were significantly lower than those of WT-BMMSCs, type 1 collagen (COL1) gene expression level was significantly lower than that of WT-BMMSCs (P<0.01), and Runt-related transcription factor-2 (RUNX2) protein expression level was significantly higher than that of WT-BMMSCs (P<0.001). While miR-145-5p and SEMA3A were up-regulated during the osteogenic differentiation of WT-BMMSCs, miR-145-5p and SEMA3A were down-regulated during the osteogenic differentiation of GK-BMMSCs, and the expression level of β-catenin (P<0.001) was considerably reduced in GK-BMMSCs. On day 21 of osteogenesis induction, WT BMMSCs had darker staining of the mineralized matrix.
Conclusion The proliferation, colony formation and osteogenic differentiation of BMMSCs decrease in type 2 diabetic rats. It is speculated that miR-145-5p plays an inhibitory role in osteogenic differentiation of BMMSCs in normal rats while a promoting role in osteogenic differentiation of BMMSCs in type 2 diabetic rats.
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