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Mariko Umeda, Fumie TERAO, Keigo Yoshizaki, Ichiro Takahashi, Profiling MicroRNA Expression in Mouse Mandibular Condylar Cartilage during Development , International Association for Dental Research, 2013.03, Objective: MicroRNA (miRNA) is one of the non-cording small RNA molecules playing important roles in development, differentiation and proliferation in various kinds of cells and organs. Mandibular condylar cartilage (MCC) is an important growth cite of mandible and face. The purpose of the present study is to identify the miRNA playing a critical role in development of MCC, and to elucidate the role of identified miRNA on chondrogenesis in MCC.
Method: Microarray analysis was performed to profile mRNAs and miRNAs among the developing MCCs isolated from embryonic day 14, 16 and 18 ICR mice. An inhibitor or mimic of miR-200a was transfected into organ culture of MCC isolated from E14 by using electroporation. After transfection, cultured explants were fixed in 4% paraformaldehyde and examined with immunohistochemistry for types I, II, and X collagens and proliferating cell nuclear antigen (PCNA). Total RNAs were isolated and analyzed for the expression of chondrogenic markers and miR-200a by quantitative RT-PCR. Cell proliferation assay was carried out by using Cell counting Kit-8, after transfection of an inhibitor or mimic for miR-200a into monolayer cell culture derived from MCC.
Result: The expression of miR-200a decreased during MCC development. The area of hypertrophic cell layer with type X collagen was expanded when miRNA-200a was inhibited. On the contrary, an area reacting to typeⅠcollagen antibody was found in the center of condylar explants, where miR-200a mimic was transfected. The number of PCNA positive cells significantly increased when the mimic is transfected (P
Conclusion: The present results suggested that miR-200a promoted proliferation, while inhibited the chondrogenic differentiation of MCC. The present research has been supported by grant-in-aid (No. 22659376) Ministry of Education, Culture, Sports, Science and Technology, Japan. . |
