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Journal of Molecular Medicine

, Volume 96, Issue 5, pp 427–444 | Cite as

Identification of novel microRNA inhibiting actin cytoskeletal rearrangement thereby suppressing osteoblast differentiation

  • Aijaz A. John
  • Ravi Prakash
  • Jyoti Kureel
  • Divya Singh
Original Article

Abstract

We report the role of miR-1187 in regulation of osteoblast functions. Over-expression of miR-1187 inhibited osteoblast differentiation. Target prediction analysis tools and experimental validation by luciferase 3′ UTR reporter assay identified BMPR-II and ArhGEF-9 as direct targets of miR-1187. ArhGEF-9 activates Cdc42 which has a major role in actin reorganization. BMP-2 also induces actin polymerization. Role of miR-1187 in actin reorganization was determined by western blotting, immunofluorescence, and in vivo gene silencing studies. Reduced protein levels of BMPR-II, activated Cdc42, and downstream signaling molecules were observed in miR-1187-transfected osteoblasts. miR-1187 over-expression resulted in decreased actin polymerization. Additionally, P-cofilin, which does not bind F-actin, was decreased in miR-1187-transfected cells. These results were corroborated by administration of BMPR-II exogenously in miR-1187-transfected osteoblasts. Silencing of miR-1187 in neonatal mice mitigated all the inhibitory effects of miR-1187 on actin cytoskeletal rearrangement. Importantly, in vivo treatment of miR-1187 inhibitor to ovariectomized BALB/c mice led to significant improvement in trabecular bone microarchitecture. Overall, miR-1187 functions as a negative regulator of osteogenesis by repressing BMPR-II and ArhGEF-9 expression thus suppressing non-Smad BMP2/Cdc42 signaling pathway and inhibiting actin reorganization. miR-1187 functions as a negative regulator of osteogenesis by repressing BMPR-II expression, which in turn, suppresses non-Smad BMP2/Cdc42 signaling pathway, thus inhibiting actin cytoskeletal rearrangement. Silencing of miR-1187 significantly improves trabecular bone microarchitecture. As miR-1187 exerts a negative regulatory role in osteoblasts function, hence, we propose that therapeutic approaches targeting miR-1187 could be useful in enhancing the bone formation and treatment of pathological conditions of bone loss.

Keywords

MicroRNA BMPR-II BMP signaling Osteoblast differentiation Actin Cell migration 

Notes

Author contributions

Experiments were conceived and designed by DS and AAJ. AAJ performed the experiments and analyzed the data. DS wrote the paper. RP and JK assisted in the experiments and helped in the analysis of the data. All authors have read and approved the manuscript before submission. The CDRI communication number is 9641.

Funding information

Fellowship grants from the Council of Scientific and Industrial Research (AAJ and JK), University Grants Commission (RP), Government of India are acknowledged.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

109_2018_1624_Fig9_ESM.gif (212 kb)
Supplementary Fig. 1

miR-1187 inhibits the expression of markers related to osteogenesis. a qRT-PCR analysis of osteoblast marker genes (Runx-2, BMP-2, type 1 collagen and Osterix) normalized to GAPDH at 48 h. miC, miR-1187, and anti-miR-1187 were transfected in mice osteoblast at 50-nM conc. b Western blot analysis of Runx-2, type I col, and BMP-2. All values represent means ± S.E. (n = 6). * P < 0.05, **P < 0.01, ***P < 0.001 compared with the control. (GIF 211 kb)

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High-resolution image (TIFF 2551 kb)
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Supplementary Fig. 2

Human osteoblasts were transfected with miC, miR-1187, and anti-miR-1187 for 48 h in differential media. a ALP activity in human osteoblasts was measured. bd qRT-PCR analysis of BMPRII and osteoblast marker genes (type 1 collagen, RUNX2) normalized to GAPDH at 48 h. All values represent means ± S.E. (n = 6). * P < 0.05, **P < 0.01, ***P < 0.001 compared with the miC (GIF 87 kb)

109_2018_1624_MOESM2_ESM.tif (1.1 mb)
High-resolution image (TIFF 1076 kb)
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Supplementary Fig. 3

miR-1187 treatment leads to sparse and thinning trabeculae, with loss of connectivity in femur bones, while anti-miR-1187 treatment significantly protected trabecular bone and exhibited greater connectivity compared to miC-treated mice. Histological analysis by H&E staining in a sham at 10× and 20×, b miC at 10× and 20×, c miR-1187 at 10× and 20×, d anti-miR-1187 at 10× and 20× (GIF 318 kb)

109_2018_1624_MOESM3_ESM.tif (4.4 mb)
High-resolution image (TIFF 4523 kb)
109_2018_1624_MOESM4_ESM.docx (13 kb)
Supplementary Table 1 (DOCX 12 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Aijaz A. John
    • 1
  • Ravi Prakash
    • 1
  • Jyoti Kureel
    • 1
  • Divya Singh
    • 1
  1. 1.Division of Endocrinology and Centre for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI)CSIR-Central Drug Research InstituteLucknowIndia

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