ABSTRACT
Osteoporosis is a metabolic disorder characterized by low bone mass and deteriorated microarchitecture, with an increased risk of fracture. Some miRNAs have been confirmed as potential modulators of osteoblast differentiation to maintain bone mass. Our miRNA sequencing results showed that miR-664-3p was significantly down-regulated during the osteogenic differentiation of the preosteoblast MC3T3-E1 cells. However, whether miR-664-3p has an impact on bone homeostasis remains unknown. In this study, we identified overexpression of miR-664-3p inhibited the osteoblast activity and matrix mineralization in vitro. Osteoblastic miR-664-3p transgenic mice exhibited reduced bone mass due to suppressed osteoblast function. Target prediction analysis and experimental validation confirmed Smad4 and Osterix (Osx) are the direct targets of miR-664-3p. Furthermore, specific inhibition of miR-664-3p by subperiosteal injection with miR-664-3p antagomir protected against ovariectomy-induced bone loss. In addition, miR-664-3p expression was markedly higher in the serum from patients with osteoporosis compared to that from normal subjects. Taken together, this study revealed that miR-664-3p suppressed osteogenesis and bone formation via targeting Smad4 and Osx. It also highlights the potential of miR-664-3p as a novel diagnostic and therapeutic target for osteoporotic patients.
Subject(s)
Cell Differentiation , MicroRNAs/genetics , Osteoblasts/pathology , Osteogenesis , Osteoporosis/pathology , Smad4 Protein/antagonists & inhibitors , Sp7 Transcription Factor/antagonists & inhibitors , Animals , Bone Density , Cell Proliferation , Cells, Cultured , Female , Gene Expression Regulation , Humans , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/pathology , Mice , Mice, Inbred C57BL , Osteoblasts/metabolism , Osteoporosis/etiology , Osteoporosis/metabolism , Smad4 Protein/genetics , Smad4 Protein/metabolism , Sp7 Transcription Factor/genetics , Sp7 Transcription Factor/metabolismABSTRACT
PURPOSE: The aim of this study was to investigate the mechanism of let-7a-5p in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in postmenopausal osteoporosis (PMOP) mice. METHODS: A mouse model of PMOP was established and osteoporosis model was identified by micro-CT scan. BMSCs in the sham group and PMOP group were cultured and osteogenic differentiation was induced. The expression of let-7a-5p in BMSCs was detected by qRT-PCR, and BMSCs was induced by osteogenic differentiation in sham and PMOP group. The BMSCs treated by let-7a-5p mimics, let-7a-5p inhibitor and negative control were named as let-7a-5p mimics group, mimics NC group, let-7a-5p inhibitor group and inhibitor NC group, respectively. ALP staining and alizarin red staining were used to detect osteogenic differentiation ability, qRT-PCR and western blot were used to detect the expression of Runt-related transcription factor 2 (Runx2) and Osterix. The targeting relationship between let-7a-5p and TGFBR1 were verificated by target scan and luciferase reporter gene assay. RESULTS: The PMOP mouse model was successfully established. The expression of let-7a-5p in BMSCs of PMOP group was significantly higher than that in the sham group (Pâ¯<â¯0.05). Let-7a-5p reduced the expression of ALP and the formation of calcified nodules, while also inhibited the expression of Runx2 and Osterix. TGFBR1 is the target gene of let-7a-5p. CONCLUSION: Let-7a-5p might inhibit the osteogenic differentiation of BMSCs in PMOP mice by regulating TGFBR1.
Subject(s)
Mesenchymal Stem Cells/cytology , MicroRNAs/physiology , Osteogenesis/drug effects , Osteoporosis, Postmenopausal/prevention & control , Receptor, Transforming Growth Factor-beta Type I/antagonists & inhibitors , Animals , Cell Differentiation , Core Binding Factor Alpha 1 Subunit/antagonists & inhibitors , Disease Models, Animal , Humans , Mesenchymal Stem Cells/drug effects , Mice , MicroRNAs/pharmacology , Sp7 Transcription Factor/antagonists & inhibitorsABSTRACT
BACKGROUND/AIMS: Osterix (Osx), a key regulator of osteoblast differentiation and bone formation, has been recently reported to be associated with the progression of breast cancer. However, the precise roles of Osx in breast cancer remain unclear. METHODS: Drug sensitivity of the cancer cells was assessed using an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Target genes were obtained by high-throughput Illumina sequencing and were confirmed in vitro and in vivo. Apoptosis was analysed by Hoechst staining and western blotting. A tissue microarray including 129 samples from breast cancer patients was used for immunohistochemistry (IHC) assays. RESULTS: Overexpression of Osx decreased the chemosensitivity of breast cancer cells, while knockdown of Osx increased the chemosensitivity of breast cancer cells. In particular, we found that the decreased chemosensitivity effect was significantly associated with elevated expression of the polypeptide N-acetylgalactosaminyltransferase 14 (GALNT14). Silencing of GALNT14 in Osx-overexpressed cells restored the decreased chemosensitivity. Conversely, overexpression of GALNT14 in Osx-knockdown cells abrogated the increased chemosensitivity in breast cancer cells. In addition, we revealed that Osx decreased GALNT14-dependent chemosensitivity by enhancing anti-apoptosis. GALNT14 expression exhibited a significant association with breast cancer stages as well as the disease-free survival (DFS) rate. CONCLUSION: Osx plays an important role in the chemosensitivity and inhibition of Osx expression may represent a therapeutic strategy to enhance the chemosensitivity of breast cancer.
Subject(s)
Breast Neoplasms/pathology , N-Acetylgalactosaminyltransferases/metabolism , Sp7 Transcription Factor/metabolism , Animals , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/toxicity , Apoptosis/drug effects , Breast Neoplasms/drug therapy , Breast Neoplasms/mortality , Cell Line, Tumor , Disease-Free Survival , Female , Humans , Immunohistochemistry , Mice , Mice, Nude , N-Acetylgalactosaminyltransferases/antagonists & inhibitors , N-Acetylgalactosaminyltransferases/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Sp7 Transcription Factor/antagonists & inhibitors , Sp7 Transcription Factor/genetics , Survival Rate , Transplantation, Heterologous , bcl-2-Associated X Protein/metabolismABSTRACT
Osteoblast differentiation is achieved by activating a transcriptional network in which Dlx5, Runx2 and Osx/SP7 have fundamental roles. The tumour suppressor p53 exerts a repressive effect on bone development and remodelling through an unknown mechanism that inhibits the osteoblast differentiation programme. Here we report a physical and functional interaction between Osx and p53 gene products. Physical interaction was found between overexpressed proteins and involved a region adjacent to the OSX zinc fingers and the DNA-binding domain of p53. This interaction results in a p53-mediated repression of OSX transcriptional activity leading to a downregulation of the osteogenic programme. Moreover, we show that p53 is also able to repress key osteoblastic genes in Runx2-deficient osteoblasts. The ability of p53 to suppress osteogenesis is independent of its DNA recognition ability but requires a native conformation of p53, as a conformational missense mutant failed to inhibit OSX. Our data further demonstrates that p53 inhibits OSX binding to their responsive Sp1/GC-rich sites in the promoters of their osteogenic target genes, such as IBSP or COL1A1. Moreover, p53 interaction to OSX sequesters OSX from binding to DLX5. This competition blocks the ability of OSX to act as a cofactor of DLX5 to activate homeodomain-containing promoters. Altogether, our data support a model wherein p53 represses OSX-DNA binding and DLX5-OSX interaction, and thereby deregulates the osteogenic transcriptional network. This mechanism might have relevant roles in bone pathologies associated to osteosarcomas and ageing.
