Abstract
BackgroundThe runt-related transcription factor 2 (Runx2) is a cell-fate-determining factor that controls osteoblast differentiation and bone formation. It has been previously demonstrated that microRNAs (miRNAs) play important roles in osteogenesis. However, the Runx2-regulated miRNAs that have been reported thus far are limited. In this study, we pursued to identify these miRNAs in Tet-on stable C2C12 cell line (C2C12/Runx2Dox subline).ResultsMicroarray analysis revealed that alterations in miRNA expression occur with 54 miRNAs. Among these miRNAs, miR-690 was identified as a positive regulator of Runx2-induced osteogenic differentiation of C2C12 cells through gain- and loss-of-function assays. Expression of miR-690 is induced by Runx2, which binds directly to the putative promoter of mir-690 (Mirn690). The miR-690 proceeds to inhibit translation of the messenger RNA encoding the nuclear factor kappa B (NF-κB) subunit p65 whose overexpression inhibits Runx2-induced osteogenic differentiation of C2C12 cells. Interleukin-6 (IL-6), a downstream target of NF-κB pathway, is upregulated by p65 overexpression but significantly downregulated during this differentiation process. Furthermore, overexpression of IL-6 impedes the expression of osteocalcin, a defined marker of late osteoblast differentiation.ConclusionsTogether, our results suggest that the miR-690 transactivated by Runx2 acts as a positive regulator of Runx2-induced osteogenic differentiation by inactivating the NF-κB pathway via the downregulation of the subunit p65.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-016-0073-y) contains supplementary material, which is available to authorized users.
Highlights
The runt-related transcription factor 2 (Runx2) is a cell-fate-determining factor that controls osteoblast differentiation and bone formation
Our findings suggest that Runx2 promotes osteogenic differentiation of C2C12 cells by inactivating nuclear factor kappa B (NF-κB) pathway via the upregulation of miR-690
In the absence of Dox, the C2C12/Runx2Dox cells cultured in medium containing 2 % horse serum can be induced into multinucleated myotubes (Additional file 1: Figure S1A)
Summary
The runt-related transcription factor 2 (Runx2) is a cell-fate-determining factor that controls osteoblast differentiation and bone formation. MicroRNAs (miRNAs) are small (approximately 22nt), functional, highly conserved, endogenous noncoding RNA They regulate protein translation or mRNA stability by imperfect binding to the 3′ untranslated region (3′UTR) of their target genes. MiR-204 and its homolog miR-211 stimulate adipocyte differentiation but inhibit osteoblast differentiation of bone marrow stromal cells through targeting runt-related transcription factor 2 (Runx2) [7]. A panel of 11 miRNAs, including miR-204, has been found to control osteogenic lineage progression by targeting Runx2 [8] These results suggest that the expression level of osteogenesis-related factors could be tightly controlled by miRNAs, which is an effective method for regulating osteogenesis. The miRNAs controlled by these factors (especially the osteoblast-specific transcription factors) are indispensable for regulating osteogenesis
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