Figure 3.
LINC00657 promoted osteogenic differentiation of T2DM-BMSCs by sponging miR-204-5p. (A) T2DM-BMSCs were transfected with the LINC00657 overexpression vector, and qPCR was performed to detect LINC00657 expression. (B) Volcano plot of DEGs in LINC00657-overexpressed T2DM-BMSCs. (C) GO enrichment of all DEGs was analyzed, and 20 osteogenically relevant GO terms were screened and presented in a bubble chart. (D) Heatmap of 40 DEGs enriched in osteogenic relevant GO terms screened in T2DM-BMSCs. T: T2DM group, C: CON group. (E) ceRNA network of LINC00657 and the top 10 DEGs. (F, G) qPCR analysis of the expression levels of miRNAs (F) and DEGs (G) in T2DM-BMSCs overexpressed LINC00657. (H) T2DM-BMSCs were transfected with the LINC00657 overexpression vector or co-transfected with miR-204-5p mimics, and qPCR was performed to detect the expression levels of miR-204-5p. (I) Dual-luciferase reporter assay was performed to detect the binding capacity of LINC00657 to miR-204-5p. (J–M) T2DM-BMSCs overexpressing LINC00657 (and miR-204-5p) were induced for 7 days, osteogenic genes (RUNX2, COL1A1, and BGLAP) were detected using qPCR (J) and western blotting (K), ALP activity was detected by staining and activity quantification (L), and mineralization level was detected using ARS staining after 14 days of induction (M). ARS, Alizarin Red staining; DEGs, differentially expressed genes; BMSCs, bone marrow-derived mesenchymal stem cells; T2DM, type 2 diabetes mellitus. *, P<0.05.
