title: MicroRNA expression profiling of human bone marrow mesenchymal stem cells during osteogenic differentiation reveals Osterix regulation by miR-31.
abstract: Osteogenesis is the result of a complex sequence of events that involve the differentiation of mesenchymal stem cells (MSC) into osteoblasts. MSCs are multipotent adult stem cells that can give rise to different cell types of the mesenchymal germ layer. The differentiation fate of MSCs depends on the microenvironmental signals received by these cells and is tightly regulated by multiple pathways that lead to the activation of specific transcription factors. Among the transcription factors involved in osteogenic differentiation Osterix (Sp7) plays a key role and has been shown to be fundamental for bone homeostasis. However, the molecular events governing the expression of this transcription factor are not fully understood. In this study we set out to investigate the changes in the microRNA (miRNA) expression that occur during the osteogenic differentiation of bone marrow-derived MSCs. To this purpose, we analyzed the miRNA expression profile of MSCs deriving from 3 donors during the differentiation and mineralization processes by microarray. 29 miRNAs were significantly and consistently modulated during the osteogenic differentiation and 5 during the mineralization process. Interestingly, most of the differentially expressed miRNAs have been reported to be implicated in stemness maintenance, differentiation and/or oncogenesis. Subsequently, we focused our attention on the regulation of Osterix by miRNAs and demonstrated that one of the miRNAs differentially modulated during osteogenic differentiation, miR-31, controls Osterix expression through association to the 3' untranslated region of this transcription factor. By analyzing miR-31 and Osterix expression levels we found an inverse miRNA-target expression trend during osteogenic differentiation and in osteosarcoma cell lines. Moreover, the inhibition of the microRNA activity led to an increase in the endogenous expression of Osterix. Our results define a miRNA signature characterizing the osteogenic differentiation of MSCs and provide evidence for the involvement of miR-31 in the regulation of the bone-specific transcription factor Osterix.
authors.affiliation: Laboratory for Orthopedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, via di Barbiano 1/10, 40136, Bologna Italy. rubinabaglio@gmail.com
authors.forename: Serena Rubina
authors.initials: SR
authors.lastname: Baglìo
authors.forename: Valentina
authors.initials: V
authors.lastname: Devescovi
authors.forename: Donatella
authors.initials: D
authors.lastname: Granchi
authors.forename: Nicola
authors.initials: N
authors.lastname: Baldini
mesh_terms: D020413:3' Untranslated Regions; D000943:Antigens, Differentiation / Q000378:metabolism; D001854:Bone Marrow Cells / Q000502:physiology; D002454:Cell Differentiation; D020869:Gene Expression Profiling; D006801:Humans; D000090062:Kruppel-Like Factor 4; D061986:MCF-7 Cells; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D035683:MicroRNAs / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D010012:Osteogenesis; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D059467:Transcriptome
chemical_list: D020413:3' Untranslated Regions; D000943:Antigens, Differentiation; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; C534612:MIRN31 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D000076182:Sp7 Transcription Factor; C478163:SP7 protein, human; D014157:Transcription Factors
keywords: 3′ untranslated region; 3′UTR; ALP; AP-1; ATF4; Activating transcription factor 4; Activator protein 1; Alkaline phosphatase; BGLAP; BMP; Bone gamma-carboxyglutamate (gla) protein; Bone morphogenetic protein; COL1A1; Collagen, type I, alpha 1; FBS; FGF; Fetal bovine serum; Fibroblast growth factor; GAPDH; Glyceraldehyde-3-phosphate dehydrogenase; KLF4; Kruppel-like factor 4; MD1; MD4; MM2; MSC; MSC differentiation 1; MSC differentiation 4; MSC mineralization 2; Mesenchymal stem cells; MiR-Vec; NFATc1; Nuclear factor of activated T-cells, cytoplasmic; Oct4; Octamer-binding transcription factor 4; Osteogenesis; Osterix miR-31 mutant sensor luciferase vector; Osterix sensor luciferase vector; Osterix-Sen-Luc; Osterix-mut31-Sen-Luc; RFU; Relative fluorescence units; Runt related transcription factor 2; Runx2; SRY (sex determining region Y)-box 2; SRY (sex determining region Y)-box 9; Sen-Luc; Sensor luciferase vector; Sox2; Sox9; Sp/XKLF; Sp7; Sp7 transcription factor (Osterix); T-cell factor/lymphoid enhancing factor; TCF/LEF; TGF-β; Telomerase RNA component; Transcription factor; Transforming growth factor, beta; Twist; Twist basic helix–loop–helix transcription factor 1; Wingless signaling pathway; Wnt; hTR; miRNA; microRNA; microRNA expressing vector; specificity protein/X Krüppel-like factor