Figure 4.

Knockdown of lncRNA-ORLNC1 Increases Osteoblast Maturation and Decreases Adipogenesis of BMSCs

(A and B) ARS (A) and ALP (B) stainings and quantitative analysis (A and B) of matrix mineralization showed BMSCs treated with shRNA-ORLNC1 increased the osteogenic ability compared with BMSCs pretreated with NC after osteogenic induction for 14 days. Scale bar, 100 μm. (C) Real-time qPCR analysis showed the increased levels of osteogenic markers in BMSCs with treatment of shRNA-ORLNC1 compared with NC as indicated. (D) Immunofluorescence staining for osteogenic protein Runx2 in BMSCs induced into osteoblasts for 3 days after transfection with shRNA-ORLNC1, normalized to the negative control group. Runx2, red; DAPI, blue. Scale bar, 100 μm. (E) Western blot analysis showed the increased levels of osteoblast genes in BMSCs after treatment with shRNA-ORLNC1. (F) ORO staining was performed to determine the number of adipocytes in BMSCs after adipogenic differentiation of 16 days. Scale bar, 200 μm. (G) The mRNA levels of adipogenesis-related genes were attenuated in the presence of shRNA-ORLNC1, as determined by real-time qPCR. (H and I) Immunofluorescence staining (H) and western blot (I) analysis indicated that the protein expression of adipocyte-specific genes were inhibited by shRNA-ORLNC1 treatment. Pparg, green; DAPI, blue. Scale bar, 100 μm. (J) H&E-stained sections of heterotopic bone showed the increased bone formation after treatment with shRNA-ORLNC1. The black arrows indicate the bone formation, and the red arrows indicate hydroxyapatite. Scale bar, 50 μm. *p < 0.05, **p < 0.01, and ***p < 0.001.