Research Paper Volume 13, Issue 14 pp 18257—18273

The lncRNA H19/miR-541-3p/Wnt/β-catenin axis plays a vital role in melatonin-mediated osteogenic differentiation of bone marrow mesenchymal stem cells

MT inhibited the adipogenic differentiation and facilitated the osteogenic differentiation of BMSCs. BMSCs were treated with 100 μM MT and/or MT2 selective inhibitor 4-P-PDOT (1 μg/ml). (A) BMSCs were cultured in adipogenic differentiation culture medium. The adipogenic differentiation of BMSCs was tested by ORO staining. Scale bar: 200 μm. (B) The expression of adipocyte-related proteins (including CEBPA, CEBPB, CEBPD, FABP4, and PPARG) after MT/4-P-PDOT treatment in BMSCs was analyzed by WB. (C) ARS activity test was conducted to evaluated the osteogenic differentiation of BMSCs. Scale: 200 μm. (D) The ALP activity was detected using ALP activity test kit. (E) The relative expression of osteogenic proteins (including ALP, BMP2, OCN, OPN and Runx2) was analyzed by WB. (F–I) The relative expression of H19 and miR-541-3p in BMSCs was analyzed by qRT-PCR. (J, K) WB was conducted to analyze the levels of APN and Wnt/β-catenin after MT treated BMSCs. *PPPP>0.05, &&PPP>0.05, ##PP

Figure 2. MT inhibited the adipogenic differentiation and facilitated the osteogenic differentiation of BMSCs. BMSCs were treated with 100 μM MT and/or MT2 selective inhibitor 4-P-PDOT (1 μg/ml). (A) BMSCs were cultured in adipogenic differentiation culture medium. The adipogenic differentiation of BMSCs was tested by ORO staining. Scale bar: 200 μm. (B) The expression of adipocyte-related proteins (including CEBPA, CEBPB, CEBPD, FABP4, and PPARG) after MT/4-P-PDOT treatment in BMSCs was analyzed by WB. (C) ARS activity test was conducted to evaluated the osteogenic differentiation of BMSCs. Scale: 200 μm. (D) The ALP activity was detected using ALP activity test kit. (E) The relative expression of osteogenic proteins (including ALP, BMP2, OCN, OPN and Runx2) was analyzed by WB. (FI) The relative expression of H19 and miR-541-3p in BMSCs was analyzed by qRT-PCR. (J, K) WB was conducted to analyze the levels of APN and Wnt/β-catenin after MT treated BMSCs. *P<0.05, **P<0.01, ***P<0.001 (vs. Con group), &P>0.05, &&P<0.01, &&&P<0.001 (vs. OS group). #P>0.05, ##P<0.01, ### P < 0.001 (vs. Adipogenic+MT group). Data were presented as mean ±SEM (n =3) and analyzed using one-way analysis of variance.