miR-23b targets Smad 3 and ameliorates the LPS-inhibited osteogenic differentiation in preosteoblast MC3T3-E1 cells

Abstract

-Lipopolysaccharide (LPS) has been confirmed to be the main inhibitor in osteogenic differentiation, posing a clinical challenge to bone healing, particularly for trauma followed by endotoxinemia/sepsis. However, the molecular mechanism remains ambiguous. miR-23b, which regulates multiple signaling pathways in inflammation, has been shown to be deregulated by LPS. In this study, we examined the LPS-mediated regulation on the expression of miR-23b and Smad 3 in preosteoblast MC3T3-E1 cells. Then we determined the regulation of miR-23b overexpression on the Smad 3 expression and on the LPS-mediated inhibition of bone morphogenetic protein-2 (BMP-2)-induced osteogenic differentiation. Our results demonstrated that LPS significantly downregulated the expression of miR-23b, while upregulating Smad 3 in MC3T3-E1 cells. However, the transfection with miR-23b mimics markedly downregulated the Smad 3 in both mRNA and protein levels, via the specific binding to the 3'-untranslated region (UTR) of Smad 3. Moreover, though LPS markedly downregulated the BMP-2-induced osteogenic differentiation of MC3T3-E1 cells by inhibiting the expression of alkaline phosphatase (ALP), Osteocalcin (OCN), Osteopontin (OPN) and Runt-related transcription factor 2 (RUNX2). The upregulated miR-23b reversed such downregulation of ALP, OCN, OPN and RUNX2 in the MC3T3-E1 cells which were treated both with LPS and BMP-2. In conclusion, our data indicates that miR-23b ameliorates the LPS-mediated inhibition of BMP-2-induced osteogenic differentiation in MC3T3-E1 cells, implying the protective role of miR-23b in the LPS-mediated inhibition of osteogenic differentiation and bone formation.

Resveratrol Attenuates Lipopolysaccharides (LPS)-Induced Inhibition of Osteoblast Differentiation in MC3T3-E1 Cells

“…Before treatment, cells were planted in tissue culture-treated plates (Corning, China) and grown to 85% confluence. To examine the effect of LPS on mitochondrial function, a variety of concentrations of LPS were used to incubate the cells (0, 0.5, 1, and 2 μg/ml) for 24 h according to a previously described method [ 19 ]. To study the effects of RSV on LPS-influenced mitochondrial function, cells were incubated for 24 h with LPS alone, LPS + RSV, or vehicle solution according to a previously published method with minor modifications [ 12 ].…”

“…The optical density (OD) values at 450 nm were detected using a fluorescence microplate reader (Bio-Rad, Hercules, CA). The aforementioned cellular levels were finally calculated according to the OD values obtained [ 19 ].…”

The Role of MicroRNAs in Bone Metabolism and Disease

“…In MC3T3-E1 cells, LPS significantly reduced the expression of miR-23b, whereas it upregulated Smad 3. Overexpression of miR-23b was demonstrated to target Smad 3, which had a negative effect on osteogenesis of MC3T3-E1 during LPS treatment [96]. The differentiated osteoblasts can have a high expression of miR-15b.…”

Dietary exosome-miR-23b may be a novel therapeutic measure for preventing Kashin-Beck disease (Review)

“…miR-23b was identified to promote the differentiation of human mesenchymal stem cells (hMSCs) into chondrocytes ( 33 ). It also depressed the expression of Smad3 at the gene and protein level, which ameliorated lipopolysaccharide-induced inhibition of osteogenic differentiation in MC3T3-E1 pre-osteoblast cells ( 34 ).…”