MicroRNA-467g inhibits new bone regeneration by targeting Ihh/Runx-2 signaling☆
Introduction
MicroRNAs (miRNAs) are small (∼22 nucleotides), single-stranded non-coding RNAs found in diverse organisms which have emerged as important post transcriptional regulators of gene expression (Dong et al., 2013, Eskildsen et al., 2011, Hassan et al., 2012, Inose et al., 2009, Li et al., 2009). MiRNAs negatively regulate translation of specific mRNAs by base pairing with partially or fully complementary sequences in target mRNAs and play a key role in various biological processes (Dong et al., 2013, Eskildsen et al., 2011, Hassan et al., 2012, Inose et al., 2009, Li et al., 2009). The expression of miRNAs is altered in various diseases like cancer, hepatitis C infection, myocardial infarction, and metabolic disease. For instance, miR-17–192 is significantly over expressed in lung cancer while let-7 is a tumor suppressor miRNA and aberrant expression of let-7 results in oncogenic loss of differentiation (Christopher et al., 2016). MiRs also regulate several properties of cardiac physiology. The examples include miR-29, miR-30 and miR-133 which are down regulated in atrial fibrillation. On the other hand, miR-328 and miR-499 are up regulated in atrial fibrillation (Santulli et al., 2014). MiR-21 is substantially increased in response to cardiac injury in experimental murine models. Inhibition of miR-21 exhibits antihypertrophic and antifibrotic effects, which leads to a significant functional improvement (Thum et al., 2008). Other examples include miR-130a which is up regulated in hepatitis C virus infection. Introduction of anti-miR-130a in hepatocytes increased IFITM1 expression with concomitantly reduction in HCV replication (Shrivastava et al., 2013).
Many miRNAs have been identified which either negatively regulate osteoblast differentiation or bone formation by targeting osteogenic factors or positively by targeting negative regulators of osteogenesis (Bae et al., 2012, Dong et al., 2013, Eskildsen et al., 2011, Gao et al., 2011, Hassan et al., 2012, Inose et al., 2009, Li et al., 2009). These include miRNAs such as 133 and 204/211 which attenuate osteoblast differentiation by directly targeting Runx2 in C2C12 mesenchymal progenitor cells and MSCs respectively (Huang et al., 2010, Li et al., 2008) or miR-141 and miR-200a which target Dlx5 to inhibit osteoblast differentiation (Itoh et al., 2009). On the contrary, miR-335-5p directly targets and down-regulates Wnt inhibitor DKK1, enhances Wnt signaling and promotes osteogenesis (Zhang et al., 2011). Besides, several microRNAs including the miR-34 family have been implicated in osteosarcoma tumorigenesis via their effects on notch signaling components (Nugent, 2015).
Previous studies by our group revealed that medicarpin, a natural pterocarpan, treatment to mice calvarial osteoblasts (MCO) led to differential regulation of several microRNAs (Kureel et al., 2014). About 80 miRNAs were down regulated by medicarpin. In the previous study we characterized the role of a well known tumor suppressor miR-542-3p in osteoblasts and found that it negatively regulated bone formation in vivo by targeting BMP-7(Kureel et al., 2014). Apart from miR-542-3p, several other candidates were robustly down regulated post medicarpin treatment and were of potential interest. In this study we report the functional characterization of miR-467g in osteoblast cells and how it suppresses new bone regeneration. Of importance was the fact, that it was a novel miRNA with no assigned role and was highly conserved across mammals like rat, chimpanzee, rhesus, humans, dog, cat and horse especially in the seed region through which miRNA recognize their target mRNAs.
Section snippets
Reagents and chemicals
Fetal bovine serum (FBS), cell culture media, α-MEM, antibiotic solution, sodium pyruvate, trypsin and non-essential amino acid were purchased from sigma (St. Louis, MO, USA). Low serum media, opti-MEM, Taqman miRNA assays, miRNA mimic, miRNA inhibitor, in vivo ready miRNA, taqman universal master mix, taqman miRNA reverse transcriptase kit and mirVana miRNA isolation kit were purchased from ambion (81 Wyman Street Waltham, MA USA). Antibodies were purchased from Abcam (Cambridge Science Park,
miR-467g is an inhibitor of osteoblast differentiation
Previous microRNA profiling results showed miR-467g to be significantly down regulated in calvarial osteoblast cells cultured in medicarpin-induced osteoblast differentiation condition (Kureel et al., 2014). This observation was validated by qRT-PCR where miR-467g expression was significantly down regulated in medicarpin and BMP2 treated mouse osteoblast cells (Fig. 1A). After confirmation by qRT-PCR data, it was decided to determine the role of miR-467g in osteoblast differentiation. Calvarial
Discussion
In the present study, we have identified a novel microRNA from osteoblasts and characterized its function. As per our knowledge, no function has been assigned to mmu-miR-467g. To determine whether miR-467g directly regulates osteoblast differentiation, we investigated the action of miR-467g in the process of osteoblastogenesis. MiR-467g over-expression inhibited osteoblast differentiation. In contrast, over expression of anti-miR-467g increased osteoblast differentiation as assessed by ALP.
Conflict of interest
The authors declare that they have no conflicts of interest with the contents of this article.
Author's contributions
JK designed and performed majority of the experiments and wrote the paper; AAJ assisted in all the experiments; MD majorly performed experiments of Fig. 5 and 6; DS conceived and designed the study and wrote the paper.
Funding source
Department of Biotechnology (DBT), Government of India and Centre for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), Council of Scientific and Industrial Research
Acknowledgements
This work was funded by Centre for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), Council of Scientific and Industrial Research and Department of Biotechnology (DBT), Government of India. JK and AAJ acknowledge CSIR, Government of India and MD acknowledge DBT, Government of India for grant of fellowship. CDRI Communication number: 9423.
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