Abstract
Osteoporosis is a frequently occurring bone disease in middle-aged and aged men and women. However, current therapies on this disease are still not ideal. MicroRNAs (miRNAs) are a class of endogenous non-protein-coding RNA with a length of 18–25 nucleotides. miRNAs have been identified as important regulators for development, metabolism, carcinogenesis, and bone formation. miR-129-5p has been reported as a regulator of cancer and neuroscience, whereas studies about its function on bone formation is still limited. In this study, we investigated the function and mechanism of miR-129-5p on osteoblast differentiation and bone formation. We have assessed the expression of miRNAs in bone mesenchymal stem cells from aging and menopause osteoporosis C57BL6 mice. The expression of miR-129-5p was altered in all osteoporosis models. Besides, the expression of miR-129-5p was negatively correlated with osteoblastic differentiation markers in the femur tissues of C57BL/6 mice of different ages. We further demonstrated that overexpression of miR-129-5p inhibited osteoblast differentiation in MC3T3-E1 cell line, as well as bone formation of C57BL/6 mice. On the other hand, down-regulation of miR-129-5p enhanced osteoblast differentiation and bone formation. We also found that miR-129-5p inhibited Wnt/β-catenin pathway in osteoblast. The target gene of miR-129-5p has been forecasted and proved as Tcf4. We further found that plasmid containing Tcf4–3′ UTR sequence enhanced osteoblast differentiation, as well as Wnt/β-catenin pathway in MC3T3-E1 cells. To further investigate the rescue effect of miR-129-5p inhibitor, we manufactured bioengineered novel recombinant miR-129-5p inhibitor through Escherichia coli system and then tested its function. The results showed that the novel recombinant miR-129-5p inhibitor promoted osteoblast differentiation and greatly ameliorated menopause osteoporosis in C57BL6 mice. In conclusion, we have discovered miR-129-5p as an inhibitor of bone formation. miR-129-5p inhibited downstream transcription factors of Wnt/β-catenin pathway through targeting Tcf4. Moreover, novel recombinant miR-129-5p inhibitor showed rescue effect on osteoporosis. This study has revealed a new mechanism of osteogenic differentiation and provided novel therapeutic strategies for treatment of skeletal disorders.
Highlights
RESULTSOsteoporosis is a high-incident bone disease in middle-aged and aged men and women, with symptoms identified as declined bone mass and bone strength, deteriorated bone microarchitecture, and increased risk of fracture (Hendrickx et al, 2015)
MicroRNAs are a class of endogenous non-protein-coding RNA with 18–25 nucleotides in length. miRNAs have been known as important regulators for development, metabolism, carcinogenesis, and bone formation (Bartel, 2009; Rigoutsos and Furnari, 2010; Chen et al, 2017)
One of the most essential causes of osteoporosis is the deterioration of osteoblast differentiation, which decreased bone formation and further led to osteoporosis
Summary
Osteoporosis is a high-incident bone disease in middle-aged and aged men and women, with symptoms identified as declined bone mass and bone strength, deteriorated bone microarchitecture, and increased risk of fracture (Hendrickx et al, 2015). Results showed that after mimic or inhibitor129-5p exposures, TCF7/LEF1 activities were reduced (approximately 47.4%, Figure 3E, P < 0.001) or enhanced (45.4%, Figure 3H, P < 0.01) These data demonstrated that miR-129-5p had inhibitory effect on both the expressions and the activities of the downstream transcript factors of Wnt/β-catenin pathway, which suggested that the regulation of miR-129-5p on osteoblast differentiation was probably through the inhibition of these transcription factors. After inhibitor-129-5p treatment, Tcf4UTR enhanced both expressions and activities of TCF7 and LEF1 by a minor extent than that in cells with normal miR-129-5p level (Figures 5D–F) All these results indicated that miR-1295p inhibited osteoblast differentiation and downstream transcript factors of Wnt/β-catenin pathway through targeting Tcf. The in vitro and in vivo results have proved the prospect that recombinant miR-1295p inhibitor may be used as a potential therapeutic drug for osteoporosis
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