Abstract
Tumor necrosis factor-alpha (TNF-α) promotes osteoclasts differentiation to enhance bone resorption and inhibits osteoblasts differentiation to impair bone formation, which plays a central role in the development of postmenopausal osteoporosis (PMOP). Recent studies implicated an important role of circular RNAs (circRNAs) in osteoporosis. The purpose of this study is to investigate whether circRNAs might be implicated in TNF-α-regulated osteoclasts differentiation and osteoblasts differentiation in PMOP. QRT-PCR was applied to detect expression of circRNA-circHmbox1 and miR-1247-5p in TNF-α-induced osteoclasts differentiation. Western blot, TRAP staining, alkaline phosphatase staining, alizarin red S staining, transwell and cell transfection were conducted to confirm that TNF-α inhibited osteoblasts differentiation by exosomal with low circHmbox1 expression from osteoclasts. Bioinformatics analysis and luciferase reporter revealed the mechanisms of the circHmbox1/miR-1247-5p/B cell lymphoma 6 (Bcl6) interaction. In this study, we found that the level of circRNA-circHmbox1 was obviously reduced in TNF-α-induced osteoclast formation in vivo and in vitro. CircHmbox1 could inhibit RANKL-induced osteoclasts differentiation primarily through binding to microRNA-1247-5p. TNF-α decreased osteoblasts differentiation by exosomal with low circHmbox1 expression from osteoclasts. Mechanistic studies showed that microRNA-1247-5p regulated osteoclasts differentiation and osteoblasts differentiation by targeting Bcl6, which was confirmed to play opposite roles in osteoblasts differentiation and osteoclasts differentiation. Our results provide evidence that circHmbox1-targeting miR-1247-5p is involved in the regulation of bone metabolisms by TNF-α in PMOP.
Highlights
Postmenopausal osteoporosis (PMOP) is caused by the imbalance between bone formation by osteoblasts and bone resorption by osteoclasts after estrogen deficiency, which is the most common metabolic bone disease
tartrate resistant acid phosphatase (TRAP) staining showed that the number of mature osteoclasts was significantly increased in receptor activator of nuclear factorκB ligand (RANKL) together with tumor necrosis factor-alpha (TNF-α) group compared to RANKL group (Figures 1B,C)
Circ_0000549, circ_0001209, circ_0001660, circ_0001667, circ_0001081 and circ_0000320 were found to be downregulated about two-folds, while circ_0000150 was upregulated in RANKL group. All of these circRNAs expression were further decreased in RANKL together with TNF-α group compared to RANKL group
Summary
Postmenopausal osteoporosis (PMOP) is caused by the imbalance between bone formation by osteoblasts and bone resorption by osteoclasts after estrogen deficiency, which is the most common metabolic bone disease. TNFα has been confirmed to affect bone metabolisms through promoting osteoclasts differentiation and inhibiting osteoblasts differentiation (Zhao, 2017). Exploring the molecular mechanisms that TNF-α regulates osteoblasts and osteoclasts differentiation is critical for researching and treating PMOP. Growing evidences have confirmed that miRNAs were involved in the regulation of bone turnover by TNF-α in estrogen deficiencyinduced osteoporosis (Maeda et al, 2017). It is unclear whether circRNAs are participated in TNF-α-regulated bone metabolism in PMOP
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