Abstract
Wnt signaling pathways are essential for bone formation. Previous studies showed that Wnt signaling pathways were regulated by miR-375. Thus, we aim to explore whether miR-375 could affect osteogenesis. In the present study, we investigated the roles of miR-375 and its downstream targets. Firstly, we revealed that miR-375-3p negatively modulated osteogenesis by suppressing positive regulators of osteogenesis and promoting negative regulators of osteogenesis. In addition, the results of TUNEL cell apoptosis assay showed that miR-375-3p induced MC3T3-E1 cell apoptosis. Secondly, miR-375-3p targeted low-density lipoprotein receptor-related protein 5 (LRP5), a co-receptor of the Wnt signaling pathways, and β-catenin as determined by luciferase activity assay, and it decreased the expression levels of LRP5 and β-catenin. Thirdly, the decline of protein levels of β-catenin was determined by immunocytochemistry and immunofluorescence. Finally, silence of LRP5 in osteoblast precursor cells resulted in diminished cell viability and cell proliferation as detected by WST-1-based colorimetric assay. Additionally, all the parameters including the relative bone volume from μCT measurement suggested that LRP5 knockout in mice resulted in a looser and worse-connected trabeculae. The mRNA levels of important negative modulators relating to osteogenesis increased after the functions of LRP5 were blocked in mice. Last but not least, the expression levels of LRP5 increased during the osteogenesis of MC3T3-E1, while the levels of β-catenin decreased in bone tissues from osteoporotic patients with vertebral compression fractures. In conclusion, we revealed miR-375-3p negatively regulated osteogenesis by targeting LRP5 and β-catenin. In addition, loss of functions of LRP5 damaged bone formation in vivo. Clinically, miR-375-3p and its targets might be used as diagnostic biomarkers for osteoporosis and might be also as novel therapeutic agents in osteoporosis treatment. The relevant products of miR-375-3p might be developed into molecular drugs in the future. These molecules could be used in translational medicine.
Highlights
Osteoporosis is a global and severe public health issue, which affects one-third women and one-fifth men in the worldwide [1].The pathological mechanisms of osteoporosis are not clearly understood
Our recent study showed that miR-9-5p, miR-675-5p and miR-138-5p damaged skeletal cell proliferation and differentiation [2]. miRNAs decrease the expression of genes by binding to the 30-untranslated region (30UTR) of target mRNAs
Our results showed that the expression levels of osteoblast differentiation biomarker RUNX2 declined (Fig 1A) while the negative regulators (SOST) of osteogenesis rose sharply (Fig 1B)
Summary
Osteoporosis is a global and severe public health issue, which affects one-third women and one-fifth men in the worldwide [1].The pathological mechanisms of osteoporosis are not clearly understood. It is essential to uncover new molecules that might play significant roles in the disturbed osteogenesis, which is a key process responsible for the pathogenesis of osteoporosis. MiRNAs play important roles in many diseases including some musculoskeletal disorders, such as osteoporosis. They can be used as the biomarkers and potential therapeutical targets. Substantial biological processes such as cell proliferation, differentiation and apoptosis are regulated by miRNAs and genes. Our recent study showed that miR-9-5p, miR-675-5p and miR-138-5p damaged skeletal cell proliferation and differentiation [2]. MiRNAs decrease the expression of genes by binding to the 30-untranslated region (30UTR) of target mRNAs Our recent study showed that miR-9-5p, miR-675-5p and miR-138-5p damaged skeletal cell proliferation and differentiation [2]. miRNAs decrease the expression of genes by binding to the 30-untranslated region (30UTR) of target mRNAs
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