MiR-433 mediates ERRγ-suppressed osteoblast differentiation via direct targeting to Runx2 mRNA in C3H10T1/2 cells.
OBJECTIVE
MicroRNAs (miRNA) are involved in various biological processes including cellular differentiation. However, the role of miR-433 in osteoblast differentiation remains poorly understood. The objective of this study was to investigate the effect of miR-433 on BMP2-induced osteoblast differentiation.


METHODS
The expression of mature miR-433 in cells was detected by real-time PCR. RT-PCR or real-time PCR was used to confirm the expression of osteogenic genes. For the activation or inhibition of miR-433 expression, we used a precursor form of miR-433 or anti-miR-433. Functional activity of miR-433 and Runx2 was evaluated by promoter study. Osteoblast differentiation was evaluated by analyzing alkaline phosphatase (ALP) activity.


RESULTS
ERRγ increased miR-433 expression in the mesenchymal stem cell lineage C3H10T1/2. During the BMP2-induction of osteoblastic differentiation of C3H10T1/2, ERRγ and miR433 expression decreased. In addition, during the osteoblastic differentiation, overexpression of ERRγ or miR-433 inhibited the expression of osteogenic marker genes such as Runx2 and ALP. A computer-based prediction algorithm led to the identification of three miR-433 binding sites [S1 (114-145 bp), S2 (3735-3766 bp) and S3 (3828-3860 bp)] on the 3'-UTR of Runx2 mRNA. Furthermore, miR-433 directly targeted S1 and S2, and decreased the level of Runx2 transcript. In addition, miR-433 inhibited BMP2-induced 6×OSE-Luc activities. Anti-miR-433 recovered ERRγ-suppressed Runx2 expression and ALP activity.


CONCLUSIONS
These results demonstrated that miR-433 suppressed BMP2-indcued osteoblast differentiation by decreasing the level of Runx2 transcript.
Department of Pharmacology and Dental Therapeutics, and Research Center for Biomineralization Disorders, School of Dentistry, Chonnam National University, Gwangju, 500-757, Republic of Korea. eunjungk@kribb.re.kr
Eun-Jung
EJ
Kim
In-Hong
IH
Kang
Jeong Woong
JW
Lee
Won-Gu
WG
Jang
Jeong-Tae
JT
Koh
D000469:Alkaline Phosphatase / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D011960:Receptors, Estrogen / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D050659:Core Binding Factor Alpha 1 Subunit; D017931:DNA Primers; C490916:Esrrg protein, mouse; D035683:MicroRNAs; C533108:Mirn433 microRNA, mouse; D012333:RNA, Messenger; D011960:Receptors, Estrogen; C493716:Runx2 protein, mouse; D000469:Alkaline Phosphatase