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MiR-542-3p suppresses osteoblast cell proliferation and differentiation, targets BMP-7 signaling and inhibits bone formation.

ABSTRACT: MicroRNAs (miRNAs) are short non-coding RNAs that interfere with translation of specific target mRNAs and thereby regulate diverse biological processes. Recent studies have suggested that miRNAs might have a role in osteoblast differentiation and bone formation. Here, we show that miR-542-3p, a well-characterized tumor suppressor whose downregulation is tightly associated with tumor progression via C-src-related oncogenic pathways, inhibits osteoblast proliferation and differentiation. miRNA array profiling in Medicarpin (a pterocarpan with proven bone-forming effects) induced mice calvarial osteoblast cells and further validation by quantitative real-time PCR revealed that miR-542-3p was downregulated during osteoblast differentiation. Over-expression of miR-542-3p inhibited osteoblast differentiation, whereas inhibition of miR-542-3p function by anti-miR-542-3p promoted expression of osteoblast-specific genes, alkaline phosphatase activity and matrix mineralization. Target prediction analysis tools and experimental validation by luciferase 3' UTR reporter assay identified BMP-7 (bone morphogenetic protein 7) as a direct target of miR-542-3p. It was seen that over-expression of miR-542-3p leads to repression of BMP-7 and inhibition of BMP-7/PI3K- survivin signaling. This strongly suggests that miR-542-3p suppresses osteogenic differentiation and promotes osteoblast apoptosis by repressing BMP-7 and its downstream signaling. Furthermore, silencing of miR-542-3p led to increased bone formation, bone strength and improved trabecular microarchitecture in sham and ovariectomized (Ovx) mice. Although miR-542-3p is known to be a tumor repressor, we have identified second complementary function of miR-542-3p where it inhibits BMP-7-mediated osteogenesis. Our findings suggest that pharmacological inhibition of miR-542-3p by anti-miR-542-3p could represent a therapeutic strategy for enhancing bone formation in vivo.

SUBMITTER: Kureel J

PROVIDER: S-EPMC3944264 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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miR-542-3p suppresses osteoblast cell proliferation and differentiation, targets BMP-7 signaling and inhibits bone formation.

Kureel J J Dixit M M Tyagi A M AM Mansoori M N MN Srivastava K K Raghuvanshi A A Maurya R R Trivedi R R Goel A A Singh D D

Cell death & disease 20140206

MicroRNAs (miRNAs) are short non-coding RNAs that interfere with translation of specific target mRNAs and thereby regulate diverse biological processes. Recent studies have suggested that miRNAs might have a role in osteoblast differentiation and bone formation. Here, we show that miR-542-3p, a well-characterized tumor suppressor whose downregulation is tightly associated with tumor progression via C-src-related oncogenic pathways, inhibits osteoblast proliferation and differentiation. miRNA arr ...[more]

PMID: 24503542

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Project description:BackgroundNon-coding RNA is a key epigenetic regulation factor during skeletal muscle development and postnatal growth, and miR-542-3p was reported to be conserved and highly expressed in the skeletal muscle among different species. However, its exact functions in the proliferation of muscle stem cells and myogenesis remain to be determined.MethodsTransfection of proliferative and differentiated C2C12 cells used miR-542-3p mimic and inhibitor. RT-qPCR, EdU staining, immunofluorescence staining, cell counting kit 8 (CCK-8), and Western blot were used to evaluate the proliferation and myogenic differentiation caused by miR-542-3p. The dual luciferase reporter analysis and rescued experiment of the target gene were used to reveal the molecular mechanism.ResultsThe data shows overexpression of miR-542-3p downregulation of mRNA and protein levels of proliferation marker genes, reduction of EdU+ cells, and cellular vitality. Additionally, knocking it down promoted the aforementioned phenotypes. For differentiation, the miR-542-3p gain-of-function reduced both mRNA and protein levels of myogenic genes, including MYOG, MYOD1, et al. Furthermore, immunofluorescence staining immunized by MYHC antibody showed that the myotube number, fluorescence intensity, differentiation index, and myotube fusion index all decreased in the miR-542-3p mimic group, compared with the control group. Conversely, these phenotypes exhibited an increased trend in the miR-542-3p inhibitor group. Mechanistically, phosphatase and tensin homolog (Pten) was identified as the bona fide target gene of miR-542-3p by dual luciferase reporter gene assay, si-Pten combined with miR-542-3p inhibitor treatments totally rescued the promotion of proliferation by loss-function of miR-542-3p.ConclusionsThis study indicates that miR-542-3p inhibits the proliferation and differentiation of myoblast and Pten is a dependent target gene of miR-542-3p in myoblast proliferation, but not in differentiation.

Dataset Information

MiR-542-3p suppresses osteoblast cell proliferation and differentiation, targets BMP-7 signaling and inhibits bone formation.

Publications

miR-542-3p suppresses osteoblast cell proliferation and differentiation, targets BMP-7 signaling and inhibits bone formation.

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