title: MicroRNA-203 up-regulates nitric oxide expression in temporomandibular joint chondrocytes via targeting TRPV4.
abstract: OBJECTIVE
  MicroRNAs (miRNAs) are recognised as important regulators of a variety of fundamental biologic processes. Our study was undertaken to examine the role of MicroRNA-203 (miR-203) in modulating nitric oxide (NO) expression in female Sprague-Dawley rat mandibular condylar chondrocytes (MCCs) via targeting transient receptor potential vanilloid 4 (TRPV4) and to demonstrate the possible mechanism of NO inhibition by chondroprotective factor 17β-oestradiol (E2).
  
  
  METHODS
  The expression of TRPV4 in mandibular condylar cartilage tissue and MCCs was detected by immunohistochemistry, immunofluorescence (IF), RT-PCR and Western blot, respectively. Primary SD rat MCCs were exposed to lipopolysaccharide (LPS), plus Ruthenium Red, 4α-phorbol 12,13-didecanoate (4αPDD), over-expressed miR-203 or E2 (10(-9) to 10(-6)M), the cellular supernatants were used for NO assay, miR-203 levels were measured by quantitative RT-PCR while TRPV4 expression changes were analysed by Western blot. The dual luciferase activity assay was performed to identify the target gene of miR-203.
  
  
  RESULTS
  TRPV4 and miR-203 were stably expressed in MCCs. The MCCs' expression of NO evoked by LPS could be enhanced or depressed by Ruthenium Red or 4αPDD. The dual luciferase assay suggested that TRPV4 was the direct target gene of miR-203. Over-expression of miR-203 inhibited the expression of TRPV4 and increased NO expression in MCCs. E2 inhibited NO expression by inhibition of miR-203, which was concurrent with the up-regulation of TRPV4 expression level in MCCs.
  
  
  CONCLUSIONS
  Our findings first suggested that miR-203 could up-regulate NO expression in female rat MCCs via targeting TRPV4. Moreover, the inhibition of NO by E2 might be at least in part through this mechanism.
authors.affiliation: Institute of Stomatology, Nanjing Medical University, Affiliated Hospital of Stomatology, Nanjing Medical University, 136 Hanzhong Road, Nanjing 210029, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Hu
authors.affiliation: Department of Oncology, First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhu
authors.affiliation: Institute of Stomatology, Nanjing Medical University, Affiliated Hospital of Stomatology, Nanjing Medical University, 136 Hanzhong Road, Nanjing 210029, China. Electronic address: nydwlktz@gmail.com.
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019902:Chondrocytes / Q000378:metabolism*; D005260:Female; D007150:Immunohistochemistry; D035683:MicroRNAs / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D050916:TRPV Cation Channels / Q000378:metabolism*; D013704:Temporomandibular Joint / Q000166:cytology* / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D035683:MicroRNAs; D050916:TRPV Cation Channels; C486577:Trpv4 protein, rat; D009569:Nitric Oxide