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

Downregulating H19 repressed the osteogenic effect of MT on BMSCs. BMSCs were transfected with sh-H19 or sh-NC, and then cultured in Adipogenic/OS differentiation culture medium. (A) Expression of H19 in BMSCs after transfection with H19 overexpression plasmids was detected by qRT-PCR. (B) ORO staining verified the role of H19 in adipogenic differentiation of BMSCs. Scale: 200 μm. (C, D) The expression of adipocyte-related proteins (including CEBPA, CEBPB, CEBPD, FABP4, and PPARG) in BMSCs was analyzed by WB. (E) ARS activity test was conducted to evaluated the osteogenic differentiation of BMSCs. Scale: 200 μm. (F) The ALP activity was detected using ALP activity test kit. (G) The relative expression of osteogenic proteins (including ALP, BMP2, OCN, OPN and Runx2) was analyzed by WB. (H) WB was utilized to analyze the protein levels of APN/Wnt/β-catenin in BMSCs cultured in adipogenic differentiation culture medium. *PPPP>0.05, &&PPPPP

Figure 4. Downregulating H19 repressed the osteogenic effect of MT on BMSCs. BMSCs were transfected with sh-H19 or sh-NC, and then cultured in Adipogenic/OS differentiation culture medium. (A) Expression of H19 in BMSCs after transfection with H19 overexpression plasmids was detected by qRT-PCR. (B) ORO staining verified the role of H19 in adipogenic differentiation of BMSCs. Scale: 200 μm. (C, D) The expression of adipocyte-related proteins (including CEBPA, CEBPB, CEBPD, FABP4, and PPARG) in BMSCs was analyzed by WB. (E) ARS activity test was conducted to evaluated the osteogenic differentiation of BMSCs. Scale: 200 μm. (F) The ALP activity was detected using ALP activity test kit. (G) The relative expression of osteogenic proteins (including ALP, BMP2, OCN, OPN and Runx2) was analyzed by WB. (H) 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, ***P<0.001(vs.Con group), &P>0.05, &&P<0.01, &&&P<0.001 (vs. Adipogenic/OS group), #P<0.05, ##P<0.01, ###P<0.01 (vs. Adipogenic/OS+MT+sh-NC group). Data were presented as mean ±SEM (n=3) and analyzed using one-way analysis of variance.