microRNA-132 inhibits osteogenic differentiation of periodontal ligament stem cells via GDF5 and the NF-κB signaling pathway

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Abstract

Background

Periodontal ligament stem cells (PDLSCs) could differentiate into osteoblasts and have a great prospect in treating bone diseases. microRNAs (miRs) and nuclear factor kappa-B (NF-κB) signaling pathway have proved pivotal in regulating osteogenic differentiation. This study intended to discuss the mechanism of miR-132 and NF-κB in PDLSC osteogenesis.

Methods

PDLSCs were firstly cultured, induced, and identified by detecting the surface markers and observing cell morphology. Levels of osteogenic markers alkaline phosphatase (ALP), bone morphogenetic proteins 2 (BMP2), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN), along with miR-132 expression were measured. The osteoblast activity and mineral deposition were detected by ALP and alizarin red S (ARS) stainings. The targeting relationship between miR-132 and growth differentiation factor 5 (GDF5) was verified. The gain-and loss-of-function was performed to discuss roles of miR-132 and GDF5 in osteogenic differentiation of PDLSCs. Besides, levels of NF-κB signaling pathway-related proteins were measured.

Results

In osteogenic differentiation of PDLSCs, levels of ALP, BMP2, Runx2 and OCN were upregulated while miR-132 was downregulated. Overexpressing miR-132 reduced levels of osteogenic markers, osteoblast activity, ALP and ARS intensity and the activation of NF-κB axis. GDF5 is a target of miR-132 and GDF5 overexpression reversed the inhibitory effects of overexpressed miR-132 on PDLSC osteogenesis.

Conclusion

Together, miR-132 could inhibit PDLSC osteogenesis via targeting GDF5 and activating NF-κB axis. These data provide useful information for PDLSC application in periodontal therapy.

Introduction

Periodontal diseases are the main chronic infectious diseases of the oral cavity, resulting in periodontal supporting tissue destruction and ultimately tooth loss [19]. The ultimate goal of periodontal therapy is to bring about periodontal tissue regeneration [15]. Periodontal ligament, kind of hypervascularized connective tissue, where osteoblasts are located and proliferate and migrate when tissues damaged, helps to maintain the stability of periodontal tissues by releasing stem cells or progenitor cells [12]. Periodontal ligament stem cells (PDLSCs), which are located around periodontal blood vessels and originated from periodontal ligament tissues, have the features of mesenchymal stem cells as indicated by their capacity for multilineage differentiation and periodontal tissue regeneration [33]. PDLSCs can reconstruct periodontal ligament tissues that have been damaged by periodontal diseases and can regulate T-cell immunity, which is important in osteogenic differentiation [20]. Zajdel A et al. have revealed osteogenesis induced by mesenchymal stem cells may be an important treatment for bone injury [32]. Thus, comprehending the molecular mechanisms of PDLSC osteogenesis could help the development of regenerative therapies for periodontal and bone diseases.

MicroRNAs (miRs) are implicated in regulation of gene expression during stem cell differentiation [11], and their function in mesenchymal stem cell osteogenesis has been verified [7]. As a member of miRs, miR-132 is extensively studied in neuronal differentiation, axon growth, neural migration, and plasticity in the nervous system and its dysregulation results in neural development and degenerative diseases [23]. A recent research has reported that miR-132 is downregulated during osteogenic differentiation of umbilical cord mesenchymal stem cells [30]. In this study, it found miR-132 could target growth differentiation factor 5 (GDF5) and inhibit its expression. GDF5, a member of the transforming growth factor beta (TGF-β) superfamily [22], is a major regulator of osteogenesis and bone repair, which induces bone formation by improving chondrocyte and osteocyte differentiation and provoking angiogenesis [5]. Additionally, GDF5 deficiency dramatically blocks PDLSC osteogenic differentiation [17]. Interestingly, it has previously justified that TGF-β could inhibit nuclear factor kappa-B (NF-κB) activity in normal cells, but activate its activity in cancerous cells [25]. Surprisingly, receptor activator of NF-κB ligand is abundantly expressed by activated T lymphocyte and B lymphocyte in periodontal disease and induces osteoclast bone resorption [18]. Besides, osteogenic differentiation of PDLSCs is inhibited by inflammation, which is regulated by NF-κB [4]. These findings offer a fundamental insight for the mechanism of periodontal disease and support the hypothesis that osteogenic differentiation of PDLSCs can be regulated via the co-work of miR-132, GDF5 and NF-κB.

Section snippets

Ethics statement

This study was approved and supervised by the ethics committee of the Beijing Stomotological Hospital Affiliated to Capital Medical University. All the subjects signed the informed consent.

Cell culture and mineralization induction

The healthy and intact premolars were extracted immediately from volunteers aged 18 ∼ 24 years who took physical examination in the Beijing Stomotological Hospital Affiliated to Capital Medical University, and the periodontal ligament tissues were isolated under aseptic conditions. The tissues were detached

Downregulation of miR-132 is found during PDLSC osteogenesis

To begin with, PDLSCs were cultured for mineralization induction. After that, cell morphology was observed, and levels of PDLSC surface markers and miR-132 were detected for PDLSC identification. Under the microscope, it was observed that the primary cells gradually assembled to form fibrous meshes on the 4th to 5th day of culture, and were spindle-shaped and closely adjacent. When culturing to the 3rd passage, cells grew rapidly and evenly, with relatively simple morphology in long spindle

Discussion

Inflammatory periodontal disease is very common and often overlooked by affected individuals [1]. What’s worse, there is a link between periodontitis and systemic diseases such as diabetes and cardiovascular diseases, which seriously impact patients’ quality of life [13]. Fortunately, researchers have confirmed PDLSCs with multi-lineage differentiation ability are effective in periodontitis treatment, and when combined with osteogenic differentiation-inducing proteins, the therapeutic potential

Declaration of Competing Interest

All authors declare that there is no conflict of interests in this study.

Acknowledgement

This work was supported by the National Natural Science Foundation of China Youth Fund (81500879).

References (33)

  • Z. Hu et al.

    miRNA-132-3p inhibits osteoblast differentiation by targeting Ep300 in simulated microgravity

    Sci. Rep.

    (2015)
  • C. Huang et al.

    MicroRNAs in regulation of osteogenic differentiation of mesenchymal stem cells

    Cell Tissue Res.

    (2017)
  • M. Hupkes et al.

    MicroRNA miR-378 promotes BMP2-induced osteogenic differentiation of mesenchymal progenitor cells

    BMC Mol. Biol.

    (2014)
  • K. Iwasaki et al.

    Periodontal ligament stem cells possess the characteristics of pericytes

    J. Periodontol.

    (2013)
  • A. Jeftha et al.

    Periodontitis and cardiovascular disease

    SADJ

    (2013)
  • F. Kidwai et al.

    Fibrinogen Induces RUNX2 Activity and Osteogenic Development from Human Pluripotent Stem Cells

    Stem Cells

    (2016)
  • Cited by (0)

    1

    These authors contributed equally to this work.

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