lncRNA-Gm5532 regulates osteoclast differentiation through the miR-125a-3p/TRAF6 axis

Abstract

Long noncoding RNAs (lncRNAs) are important regulators of bone metabolism. In this study, lncRNA microarray analysis was used to identify differentially expressed lncRNAs in differentiated osteoclasts. lncRNA-Gm5532 is highly expressed during osteoclast differentiation. lncRNA-Gm5532 knockdown impairs osteoclast formation and bone resorption. Mechanistic experiments show that lncRNA-Gm5532 functions as a competing endogenous RNA (ceRNA) and acts as a sponge for miR-125a-3p, which promotes TNF receptor-associated factor 6 (TRAF6) expression. miR-125a-3p mimics suppress osteoclast differentiation and TAK1/NF-κB/MAPK signaling. The miR-125a-3p inhibitor reverses the negative effects of siGm5532 on osteoclast differentiation. In summary, our study reveals that lncRNA-Gm5532 functions as an activator in osteoclast differentiation by targeting the miR-125a-3p/TRAF6 axis, making it a novel biomarker and potential therapeutic target for osteoporosis.

Keywords: TNF receptor-associated factor 6 (TRAF6); lncRNA-Gm5532; miR-125a-3p; osteoclast differentiation.

Figure 1

lncRNA-Gm5532 is highly expressed during osteoclast differentiation (A) RAW264.7 cells were differentiated into osteoclasts upon treatment with 50 ng/mL RANKL for 4 days. TRAP staining and F-actin staining were used to detect TRAP-positive multinucleated cells and ring-like actin structures. (B) Western blot analysis of the protein levels of CTSK, MMP9 and NFATc1 in RAW264.7-derived osteoclasts. (C) Volcano plot of the lncRNA expression signature in undifferentiated RAW264.7 cells and RAW264.7-derived osteoclasts. (D) The hierarchical clustering heatmap of lncRNAs that are highly expressed in RAW264.7-derived osteoclasts. (E) lncRNA-Gm5532 expression in undifferentiated RAW264.7 cells and RAW264.7-derived osteoclasts. Data are presented as the mean±SD. ***P<0.001 vs Un-Diff.

Figure 2

lncRNA-Gm5532 knockdown suppresses osteoclast formation and bone resorption (A) The siRNA method was used to downregulate lncRNA-Gm5532 expression. (B) RAW264.7 cells were transfected with siGm5532 and then incubated with 50 ng/mL RANKL. Osteoclast formation was analyzed by the area of TRAP-positive multinucleated cells. Scale bar: 200 μm. (C) Bone resorption was analyzed by the area of resorption pits. Scale bar: 200 μm. (D) TRAP activity was expressed as nanomoles of pNPP hydrolyzed per 10 min per microgram of protein. (E) Western blot analysis of the protein levels of CTSK, MMP9 and NFATc1. (F) mRNA expression of bone resorption-associated genes was measured on day 2. Data are presented as the mean±SD. *P<0.05, **P<0.01, ***P<0.001 vs NC.

Figure 3

miR-125a-3p is the target of lncRNA-Gm5532 (A) Complementary sequences of lncRNA-Gm5532 and miR-125a-3p. (B) The putative binding sites of lncRNA-Gm5532 were mutated and inserted into the psiCHECK-2 plasmid. 293T cells cotransfected with miR-125a-3p mimics and Gm5532-WT or Gm5532-MT plasmid were detected for luciferase activity. (C) miR-125a-3p expression after siGm5532 treatment during osteoclast differentiation. Data are presented as the mean±SD. *P<0.05 vs NC mimics or NC.

Figure 4

miR-125a-3p suppresses osteoclast differentiation (A) RAW264.7 cells were transfected with miR-125a-3p mimics and then incubated with 50 ng/mL RANKL. Osteoclast formation was analyzed by the area of TRAP-positive multinucleated cells. Scale bar: 200 μm. (B) Bone resorption was analyzed by the area of resorption pits. Scale bar: 200 μm. (C) miR-125a-3p expression during osteoclast differentiation. (D) TRAP activity was expressed as nanomoles of pNPP hydrolyzed per 10 min per microgram of protein. (E) Western blot analysis of the protein levels of CTSK, MMP9 and NFATc1. (F) mRNA expression of bone resorption-associated genes was measured on day 2. Data are presented as the mean±SD. *P<0.05, **P<0.01, ***P<0.001 vs NC or Un-Diff.

Figure 5

lncRNA-Gm5532 regulates osteoclast differentiation by targeting miR-125a-3p (A) RAW264.7 cells were cotransfected with siGm5532 and miR-125a-3p inhibitor. After that, the cells were incubated with 50 ng/mL RANKL. miR-125a-3p expression was detected on day 2. (B,C) Osteoclast formation was analyzed by the area of TRAP-positive multinucleated cells. Scale bar: 200 μm. (D,E) Bone resorption was analyzed by the area of resorption pits. Scale bar: 200 μm. (F) TRAP activity was expressed as nanomoles of pNPP hydrolysed per 10 min per microgram of protein. (G) Western blot analysis of the protein levels of CTSK, MMP9 and NFATc1. Data are presented as the mean±SD. *P<0.05, **P<0.01, ***P<0.001.

Figure 6

TRAF6 is the target of miR-125a-3p (A) Complementary sequences of TRAF6 and miR-125a-3p. (B) The putative binding sites of TRAF6 were mutated and inserted into the psiCHECK-2 plasmid. 293T cells cotransfected with miR-125a-3p mimics and TRAF6-WT or TRAF6-MT plasmid were detected for luciferase activity. (C) TRAF6 protein expression was detected on day 2 during osteoclast differentiation. (D) TRAF6 mRNA expression was detected on day 2 during osteoclast differentiation. (E) miR-125a-3p suppressed the mRNA expression of TRAF6 on day 2 during osteoclast differentiation. (F,G) After RAW264.7 cells were transfected with miR-125a-3p mimics, the cells were incubated with 50 ng/mL RANKL. Cell lysates were collected at 0 min, 10 min, and 20 min and immunoblotted with anti-phosphorylated and total TAK1, IKK, IκBα, p65, ERK, JNK, and p38 antibodies. *P<0.05, **P<0.01, ***P<0.001 vs NC mimic, Un-Diff, or NC.