Abstract
Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) are a potential source of cells for therapeutic applications in bone regeneration. Recent evidence reveals a role for microRNAs (miRNAs) in the fine-tuning regulation of osteogenesis (osteomiRs) suggesting that they can be potential targets for skeleton diseases treatment. However, the functions of osteomiRs during differentiation of hAM-MSCs to osteogenic lineage are poorly understood. In this investigation, we discovered a novel miRNAs expression signature corresponding to the matrix maturation (preosteoblast) and mineralization (mature osteoblast) stages of dexamethasone-induced osteoblastic differentiation of hAM-MSCs. Comprehensive miRNAs profiling using TaqMan Low Density Arrays showed that 18 miRNAs were significantly downregulated, whereas 3 were upregulated in the matrix maturation stage (7 days after osteogenic induction) in comparison to undifferentiated cells used as control. Likewise, 47 miRNAs were suppressed and 25 were overexpressed at mineralization stage (14 days after osteogenic induction) in comparison to osteoprogenitors cells. Five out 93 miRNAs (miR-19b-3p, miR-335-3p, miR-197-3p, miR-34b-39, and miR-576-3p) were regulated at both 7 and 14 days suggesting a role in coordinated guidance of osteoblastic differentiation. Exhaustive bioinformatic predictions showed that the set of modulated miRNAs may target multiple genes involved in regulatory networks driving osteogenesis including key members of BMP, TGF-β, and WNT/β-catenin signaling pathways. Of these miRNAs, we selected miR-204, a noncoding small RNA that was expressed at matrix maturation phase and downregulated at maturation stage, for further functional studies. Interestingly, gain-of-function analysis showed that restoration of miR-204 using RNA mimics at the onset of mineralization stage dramatically inhibited deposition of calcium and osteogenic maturation of hAM-MSCs. Moreover in silico analysis detected a conserved miR-204 binding site at the 3′UTR of TGF-βR2 receptor gene. Using luciferase assays we confirmed that TGF-βR2 is a downstream effector of miR-204. In conclusion, we have identified a miRNAs signature for osteoblast differentiation of hAM-MSCs. The results from this study suggested that these miRNAs may act as potential inhibitors or activators of osteogenesis. Our findings also points towards the idea that miR-204/TGF-βR2 axis has a regulatory role in differentiation of hAM-MSCs committed to osteoblastic lineage.
Highlights
Osteoporosis disease and bone fractures are among the major public health concerns worldwide, as they produce a decline in patient mobility and a significant increase in medical care costs [1]
We have isolated and characterized the biological events underlying the osteoblastic differentiation of the hAM-MSCs subpopulation studied here and established the temporality of proliferation, matrix maturation, and mineralization stages [13, 14]
Number of miRNAs 17 20 20 20 18 21 19 21 16 18 18 20 20 21 17 osteogenesis process, we focused on the set of upregulated miRNAs and found that they may attenuate a large list of key genes involved in TGF-β and WNT/β-catenin pathways which are critical for osteogenic differentiation and bone formation
Summary
Osteoporosis disease and bone fractures are among the major public health concerns worldwide, as they produce a decline in patient mobility and a significant increase in medical care costs [1]. A recent review on this subject summarize the evidence indicating that both skeletal development and bone regeneration depend on the proper functional differentiation of mesenchymal stem cells to osteoblasts [2]. Human mesenchymal stem cells are pluripotent stem cells that undergo a multistage differentiation process in which they proliferate and differentiate into osteocytes, adipocytes, and chondrocytes [3]. Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) have been used for in vivo construction of tissue-engineered cartilage, bone, and other soft tissues, and they have become a promising tool for the treatment of bone diseases [4]. Elucidating the molecular mechanisms that regulate the osteoblastic differentiation is essential to help understand the molecular mechanisms of osteogenesis, but it may be a guide for the development of novel therapies for the treatment of bone lesions and osteoporosis
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