title: Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor.
abstract: The molecular mechanisms that regulate hADSC differentiation toward osteogenic precursors and subsequent bone-forming osteoblasts is unknown. Using osteoblast precursors obtained from subcutaneous human adipose tissue, we observed that microRNA-26a modulated late osteoblasts differentiation by targeting the SMAD1 transcription factor.
  
  
  BACKGROUND
  Elucidation of the molecular mechanisms guiding human adipose tissue-derived stem cells (hADSCs) differentiation is of extreme importance for improving the treatment of bone-related diseases such as osteoporosis. The aim of this study was to identify microRNA as a regulator of the osteogenic differentiation of hADSCs.
  
  
  METHODS
  Osteoblast differentiation of hADSCs was induced by treatment with dexamethasone, ascorbic acid, and beta-glycerol phosphate. The expression of osteoblastic phenotype was evaluated after the induction by simultaneous monitoring of alkaline phosphatase activity, the expression of genes involved in osteoblastic differentiation by real-time RT-PCR, and mineralization at the same time. MicroRNA expression was determined by Northern blot, and transfection of both antisense miR-RNA and sensor plasmids was done to validate the inhibitory role of microRNA during hADSC osteogenesis. Western blot was used to determine the expression levels of the SMAD1 protein. qRT-PCR analysis was used to compare the expression patterns of osteoblastic markers in transfected cells.
  
  
  CONCLUSIONS
  We analyzed the role of microRNA 26a (miR-26a) during differentiation of hADSCs. Northern blot analysis of miR-26a during hADSC differentiation showed increased expression, whereas expression of the SMAD1 protein was complementary to that of miR-26a. Because the highest expression of miR-26a and the lowest expression of SMAD1 protein were reached at hADSC terminal differentiation, we carried out our study during the late stages of hADSC differentiation. The inhibition of miR-26a, by 2'-O-methyl-antisense RNA, increased protein levels of its predicted target, SMAD1 transcription factor, in treated osteoblasts, upregulating bone marker genes and thus enhancing osteoblast differentiation. Our data suggest a role for miR-26a in the differentiation induced by treatment with dexamethasone, ascorbic acid, and beta-glycerol phosphate of hADSCs toward the osteogenic lineage by targeting its predicted target, the SMAD1 protein. This study contributes to a better knowledge of molecular mechanisms governing hADSC differentiation by proposing a microRNA-based control of late differentiation.
authors.affiliation: Metabolic Bone Unit, Laboratory of Molecular Genetics, Department of Internal Medicine, University of Florence, Florence, Italy.
authors.forename: Ettore
authors.initials: E
authors.lastname: Luzi
authors.forename: Francesca
authors.initials: F
authors.lastname: Marini
authors.forename: Silvia Carbonell
authors.initials: SC
authors.lastname: Sala
authors.forename: Isabella
authors.initials: I
authors.lastname: Tognarini
authors.forename: Gianna
authors.initials: G
authors.lastname: Galli
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Brandi
mesh_terms: D020413:3' Untranslated Regions / Q000378:metabolism; D000273:Adipose Tissue / Q000166:cytology*; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D051898:Smad1 Protein / Q000096:biosynthesis / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*
chemical_list: D020413:3' Untranslated Regions; D035683:MicroRNAs; D012333:RNA, Messenger; D051898:Smad1 Protein; C528163:MIRN26 microRNA, human