title: Profiling microRNA expression during fracture healing.
abstract: BACKGROUND
  The discovery of microRNA (miRNA) has revealed a novel type of regulatory control for gene expression. Increasing evidence suggests that miRNA regulates chondrocyte, osteoblast, and osteoclast differentiation and function, indicating miRNA as key regulators of bone formation, resorption, remodeling, and repair. We hypothesized that the functions of certain miRNAs and changes to their expression pattern may play crucial roles during the process of fracture healing.
  
  
  METHODS
  Standard healing fractures and unhealing fractures produced by periosteal cauterization at the fracture site were created in femurs of seventy rats, with half assigned to the standard healing fracture group and half assigned to the nonunion group. At post-fracture days 3, 7, 10, 14, 21, and 28, total RNA including miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was performed with miRNA samples from each group on post-fracture day 14. For further analysis, we selected highly up-regulated five miRNAs in the standard healing fracture group from the microarray data. Real-time PCR was performed with miRNA samples at each time point above mentioned to compare the expression levels of the selected miRNAs between standard healing fractures and unhealing fractures and investigate their time-course changes.
  
  
  RESULTS
  Microarray and real-time polymerase chain reaction (PCR) analyses on day 14 revealed that five miRNAs, miR-140-3p, miR-140-5p, miR-181a-5p, miR-181d-5p, and miR-451a, were significantly highly expressed in standard healing fractures compared with unhealing fractures. Real-time PCR analysis further revealed that in standard healing fractures, the expression of all five of these miRNAs peaked on day 14 and declined thereafter.
  
  
  CONCLUSIONS
  Our results suggest that the five miRNAs identified using microarray and real-time PCR analyses may play important roles during fracture healing. These findings provide valuable information to further understand the molecular mechanism of fracture healing and may lead to the development of miRNA-based tissue engineering strategies to promote fracture healing.
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. orutop@yahoo.co.jp.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Waki
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. sangyang@beige.plala.or.jp.
authors.forename: Sang Yang
authors.initials: SY
authors.lastname: Lee
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. tniikura@med.kobe-u.ac.jp.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Niikura
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. t_iwakura1978@yahoo.co.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Iwakura
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. ydogaki@hotmail.co.jp.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Dogaki
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. agora.agora.eo@gmail.com.
authors.forename: Etsuko
authors.initials: E
authors.lastname: Okumachi
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. keisuke5091@gmail.com.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Oe
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. kurodar@med.kobe-u.ac.jp.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Kuroda
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. kurosaka@med.kobe-u.ac.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kurosaka
mesh_terms: D000818:Animals; D005265:Femoral Neck Fractures / Q000000981:diagnostic imaging / Q000235:genetics / Q000378:metabolism; D017102:Fracture Healing / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D008297:Male; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics*; D011859:Radiography; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D035683:MicroRNAs