Figure 4: miR-21 regulates receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) by targeting Sprouty 1 (Spry1) to modulate extracellular signal-regulated kinase (ERK) signaling in osteoblasts (OBs). | Scientific Reports

Figure 4: miR-21 regulates receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) by targeting Sprouty 1 (Spry1) to modulate extracellular signal-regulated kinase (ERK) signaling in osteoblasts (OBs).

From: miR-21 deficiency inhibits osteoclast function and prevents bone loss in mice

Figure 4

(a,b) Enzyme-linked immunosorbent assay (ELISA) detection of serum concentrations of RANKL (a) and OPG (b). Increased RANKL and decreased OPG were detected in miR-21−/− mice, suggest that the increased bone mass in miR-21−/− mice was not attributed to RANKL or OPG changes. (c) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated up-regulated mRNA level of RANKL and down-regulated mRNA level of OPG in OBs from 3-month WT and miR-21−/− mice. (d) Concentrations of RANKL and OPG were determined by ELISA in culture media of OBs. miR-21−/− OBs showed increased RANKL secretion and decreased OPG secretion. (e) qRT-PCR analysis of miR-21−/− OBs demonstrated down-regulation of mRNA level of SPRY1 by small interfering RNA. siSPRY1, small interfering RNA for SPRY1. NC, negative control of siSPRY1. (f) qRT-PCR analysis demonstrated suppression of mRNA level of RANKL and rescue of mRNA level of OPG in miR-21−/− OBs by siSPRY1. (g) Western blot analysis of miR-21−/− OBs. siSPRY1 stimulated ERK signaling at both total and phosphorylated protein expression levels. Cropped blots are displayed with only brightness adjusted equally across the entire images. (h) qRT-PCR analysis demonstrated increased mRNA level of RANKL and decreased mRNA level of OPG in SPRY1-down-regulated miR-21−/− OBs by PD98059, an ERK inhibitor. (i) Concentrations of RANKL and OPG were determined by ELISA in culture media of miR-21−/− OBs. Data demonstrated that miR-21 regulated RANKL and OPG by targeting Spry1 to regulate ERK signaling. Data represents mean ± standard errors of the mean. n = 6/genotype (ad), n = 3/group (eh) and n = 4/group (i). Statistical significance was evaluated by two-tailed Student’s t test for two-group comparison, and one way analysis of variation (ANOVA) with Newman-Keuls post-hoc tests for multiple comparisons. *P < 0.05. NS, not significant (P > 0.05).

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