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

H19 affected the MT-induced osteogenic and adipogenic differentiation of BMSCs by inhibiting the miR-541-3p/APN axis. H19 overexpression plasmids and miR-541-3p mimics were transfected into BMSCs, which were cultured in adipogenic/osteogenic differentiation medium and treated with MT (100 μM). (A, B) The H19 and miR-541-3p expression in adipogenic/osteogenic BMSCs was monitored by qRT-PCR. (C) ORO staining verified the role of H19 in adipogenic differentiation of BMSCs. Scale: 200 μm. (D, E) The expression of adipocyte-related proteins (including CEBPA, CEBPB, CEBPD, FABP4, and PPARG) in BMSCs was analyzed by WB. (F) ARS activity test was conducted to evaluated the osteogenic differentiation of BMSCs. Scale: 200 μm. (G) The ALP activity was detected using ALP activity test kit. (H) The relative expression of osteogenic proteins (including ALP, BMP2, OCN, OPN and Runx2) was analyzed by WB. (I) WB was utilized to analyze the protein levels of APN/Wnt/β-catenin in BMSCs cultured in adipogenic differentiation culture medium. *PPPPPPPP

Figure 7. H19 affected the MT-induced osteogenic and adipogenic differentiation of BMSCs by inhibiting the miR-541-3p/APN axis. H19 overexpression plasmids and miR-541-3p mimics were transfected into BMSCs, which were cultured in adipogenic/osteogenic differentiation medium and treated with MT (100 μM). (A, B) The H19 and miR-541-3p expression in adipogenic/osteogenic BMSCs was monitored by qRT-PCR. (C) ORO staining verified the role of H19 in adipogenic differentiation of BMSCs. Scale: 200 μm. (D, E) The expression of adipocyte-related proteins (including CEBPA, CEBPB, CEBPD, FABP4, and PPARG) in BMSCs was analyzed by WB. (F) ARS activity test was conducted to evaluated the osteogenic differentiation of BMSCs. Scale: 200 μm. (G) The ALP activity was detected using ALP activity test kit. (H) The relative expression of osteogenic proteins (including ALP, BMP2, OCN, OPN and Runx2) was analyzed by WB. (I) WB was utilized to analyze the protein levels of APN/Wnt/β-catenin in BMSCs cultured in adipogenic differentiation culture medium. *P<0.05, **P<0.01 (vs. Con group), &P<0.05, &&P<0.01, &&&P<0.001 (vs.Adipogenic/OS+MT group), #P<0.05, ##P<001, ###P<0.01 (vs. Adipogenic/OS +MT+miR-541-3p). Data were presented as mean ±SEM (n =3) and analyzed using one-way analysis of variance.