title: miR-221 attenuates the osteogenic differentiation of human annulus fibrosus cells.
abstract: In the moderate and end stages of intervertebral disc (IVD) degeneration, endochondral ossifications are found in the IVD.
  
  
  
  The aim of this study was to investigate whether endochondral ossification in the late stages of disc degeneration is due to the differentiation of resident progenitor cell in the annulus fibrosus (AF) and the potential signaling pathways in vitro.
  
  
  
  This is an in vitro study of AF cell osteogenic differentiation and possible mechanisms
  
  
  
  Normal annulus fibrosus (NAF) and degenerated annulus fibrosus (DAF) cells were isolated from tissue removed surgically from juvenile patients with idiopathic scoliosis and adult patients with degenerative scoliosis. Osteogenic differentiation was investigated using quantitative reverse transcription polymerase chain reaction (RT-PCR) and histology. The effects of miR-221 on osteogenesis were measured by overexpression of miR-221 with lentivirus. BMP2 and phospho-Smad proteins were detected by Western blotting.
  
  
  
  Both NAF and DAF cells underwent osteogenic differentiation, which was confirmed by detecting mineralization of the cell cultures and by an increase in the expression mRNAs for BMP2, runx2, alkaline phosphatase (ALP), and osteocalcin. DAF cells exhibited increased osteogenic differentiation potential over the NAF cells. By contrast to the elevated phospho-Smads, the basal level of miR-221 significantly decreased in DAF cells compared with that in NAF cells. Cultures of both cell types in osteogenic medium showed a decrease in miR-221 expression, and overexpression of miR-221 markedly decreased the level of BMP2, phospho-Smads, and the expression of osteogenic genes in DAF cells. The osteogenic potential of DAF cells diminished by the overexpression of miR-221.
  
  
  
  Compared with NAF cells, AF cells from degenerated discs have a greater tendency for osteogenic differentiation, which involves the BMP-Smad pathways and can be regulated by miR-221. These observations may be developed into a therapeutic to prevent the endochondral ossification.
authors.affiliation: Department of Orthopaedic Surgery, University of Virginia, Hospital Drive, Cobb Hall B039, Charlottesville, VA 22908, USA.
authors.forename: Ching-Hua
authors.initials: CH
authors.lastname: Yeh
authors.affiliation: Department of Orthopaedic Surgery, University of Virginia, Hospital Drive, Cobb Hall B039, Charlottesville, VA 22908, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Jin
authors.affiliation: Department of Orthopaedic Surgery, University of Virginia, Hospital Drive, Cobb Hall B039, Charlottesville, VA 22908, USA.
authors.forename: Francis
authors.initials: F
authors.lastname: Shen
authors.affiliation: Department of Orthopaedic Surgery, University of Virginia, Hospital Drive, Cobb Hall B039, Charlottesville, VA 22908, USA.
authors.forename: Gary
authors.initials: G
authors.lastname: Balian
authors.affiliation: Department of Orthopaedic Surgery, University of Virginia, Hospital Drive, Cobb Hall B039, Charlottesville, VA 22908, USA. Electronic address: lixudong@virginia.edu.
authors.forename: Xudong Joshua
authors.initials: XJ
authors.lastname: Li
mesh_terms: D000293:Adolescent; D000328:Adult; D000070616:Annulus Fibrosus / Q000166:cytology / Q000378:metabolism*; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D055959:Intervertebral Disc Degeneration / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics*; D008875:Middle Aged; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D010012:Osteogenesis; D051785:Smad Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D055396:Bone Morphogenetic Protein 2; D035683:MicroRNAs; D015675:Osteocalcin; D051785:Smad Proteins; C525718:BMP2 protein, human; C525709:MIR221, human
keywords: Annulus fibrosus; BMP; Gene therapy; Intervertebral disc; Osteogenesis; miR-221