The role of microRNA in periodontal tissue: A review of the literature

Archives of Oral Biology 72 (2016) 66–74

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Review

The role of microRNA in periodontal tissue: A review of the literature Rizky Aditiya Irwandi, Anjalee Vacharaksa* Research Unit on Oral Microbiology and Immunology, Microbiology Department, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand

A R T I C L E I N F O

Article history: Received 6 June 2016 Received in revised form 4 August 2016 Accepted 12 August 2016 Keywords: MicroRNA Bone remodeling Inflammatory mediators Periodontal tissue RANKL

A B S T R A C T

MicroRNAs (miRNAs) bind at the 30 UTR of their target mRNA to induce gene silencing. Through this mechanism, number of biological pathways implicated in developmental, physiological, and pathological processes, have been frequently found to involve miRNA functions. MiRNA functions in bone metabolism have also been reported, especially in association with receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis. Expression of RANKL has been related to several inflammatory mediators, and thus some miRNAs may be implicated in the regulatory mechanism of inflammatoryinduced RANKL expression as shown in periodontal resident cells such as gingival fibroblasts or periodontal ligament cells. This review aims to review the current miRNA research relating periodontal tissue and its relevance in periodontal inflammation. In miRNA profiling studies of tissues isolated from individuals with periodontal disease, miR-223 has been consistently identified as a potential candidate miRNA to be further investigated in periodontitis-related processes. Although these studies point to an important role of miRNA-mediated epigenetic changes in tissue inflammation and alveolar bone loss, further investigation is still required to determine the function of miRNAs in the complex processes of periodontal tissue homeostasis and pathogenesis. Knowledge gained from future studies will be beneficial in developing alternative therapeutic approaches, especially ones that use miRNA delivery systems to treat periodontal disease. ã 2016 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5.

MicroRNA biology and function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bone remodeling and the role of inflammatory mediators in periodontal tissue . The role of miRNA in bone remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MicroRNA dynamics in periodontal tissue and periodontitis-related mechanisms Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. MicroRNA biology and function Epigenetic regulation plays a role in defining the specificity of gene expression in living organisms. Approximately 0.5% of the human genome varies from person to person. Chimpanzees and humans, on the other hand, share 98% of their genomes, yet are very distinct from one another (Varki & Altheide, 2005). The phenotypic differences within and between species with very similar genomes probably result largely from epigenetic control.

* Corresponding author. E-mail address: [email protected] (A. Vacharaksa). http://dx.doi.org/10.1016/j.archoralbio.2016.08.014 0003-9969/ã 2016 Elsevier Ltd. All rights reserved.

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66 67 68 70 72 72

Each cell that makes up an organism has the ability to alter chromatin structure, which results in changes in DNA sequence read-out. Epigenetic mechanisms, including DNA methylation, histone modification, chromatin remodeling, and small interfering RNAs, are integrated in cooperative and regulatory networks to control biological changes (Murr, 2009). Studies on small RNAs, especially microRNAs (miRNAs), have been increasing substantially over the past two decades, since the discovery of miRNA in 1993 (Lee, Feinbaum, & Ambros, 1993; Almeida, Reis, & Calin, 2011). A number of biological pathways, implicated in developmental, physiological, and pathological processes, have been frequently found to involve miRNA functions (Neo et al., 2014; Lin, Yue, Pan, Sun, & Wang, 2011; Parikh et al., 2014).

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The miRNA is composed of approximately 22 nucleotides. Six of these nucleotides at the 50 end, known as ‘seed sequence’, can specifically bind to 30 UTRs of target messenger RNAs (mRNAs), leading to translational repression (Huntzinger & Izaurralde, 2011; Pasquinelli, 2012). The miRNA can cause structural disruption of mRNAs in two different ways, depending on the number of basepairing with its target mRNA. For translational blockage in plant cells, the 22-nucleotide miRNA is required to completely bind to its target mRNA (Brodersen et al., 2008), leading to the degradation of the target mRNA through the endonuclease activity of the miRNA silencing machinery. In contrast, partial pairing of the 6-nucleotide of the seed sequence is adequate to induce the de-adenylation of the target mRNA resulted in structural disruption (Pasquinelli, 2012) and translational blockage in animal cells (Bartel, 2009). In the canonical targeting pattern, 6-mer at positions 2–8 of miRNA is perfectly matched with the sequence of the transcript while the imperfect match refers to a non-canonical type (Hausser & Zavolan, 2014). The canonical type effectively causes target repression (Wang, 2014). By these mechanisms, each miRNA, not only directly represses a number of target genes, but also indirectly affects other associated genes to regulate cell functions (Baek et al., 2008; Selbach et al., 2008). Although all mechanisms can block translation, whether silencing occurs predominantly by mRNA structural disruption or by translational interference remains controversial. MiRNA biogenesis is the process by which pri-miRNAs are sequentially cleaved to first generate pre-miRNAs, which are in turn cleaved to produce the mature miRNAs. The process starts in the nucleus, where pri-miRNAs are transcribed by the RNA polymerase II. The pri-miRNA is a long primary transcript with a local stem-loop structure that contains a local hairpin structure encoding the miRNA sequence. The microprocessor complex, composed of Drosha and DiGeorge Syndrome Chromosomal Region 8 (DGCR8), subsequently cleaves the pri-miRNA to produce the pre-miRNA (Han et al., 2004). The pre-miRNA is then exported from the nucleus to the cytoplasm by exportin-5. In the cytoplasm, pre-miRNA is cleaved to become an 18–25-nucleotide-long double-stranded RNA (dsRNA) through the function of Dicer, an RNase III-type endonuclease that acts specifically on dsRNAs. One strand of this hairpin duplex is loaded into the Argonaute (AGO) protein to form the RNA-induced silencing complex (RISC). The mature miRNAs in complex with RISC are able to target mRNAs through base-pairing (Ha & Kim, 2014). MiRNAs play various roles in complex biological cascades including the inflammatory responses. The shift in miRNA profiles in the gingiva and salivary gland, for example, indicates the miRNA-related host immune response to oral bacterial infection at the primary and secondary infection sites in a rat model (Nayar et al., 2016). In autoimmune disease, increase of miR-148a enhances B cells auto-reactivity by targeting the autoimmune suppressor, Gadd45a, the tumor suppressor, PTEN and proapoptotic protein, Bim (Gonzalez-Martin et al., 2016), while anti-inflammatory effect of miR-24 is reported in macrophage (Fordham, Naqvi, & Nares, 2015). The inflammatory mediator, tumor necrosis factor (TNF)-a, activates osteoclastogenesis in mouse bone marrow-derived precursor cells through the upregulation of miR-182 (Miller et al., 2016). Thus, miR-182 inhibitor may represent an effective regulator to control inflammatory osteoclastogenesis and bone resorption. These findings emphasize the important functions of miRNAs and epigenetic regulation. This review will discuss the role of miRNAs in healthy and inflamed periodontal tissues as well as the miRNAs in inflammatory-related bone remodeling that have been reported in periodontal resident cells. The dynamics of miRNA profiles in periodontal tissue as related to periodontitis is summarized.

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2. Bone remodeling and the role of inflammatory mediators in periodontal tissue The physiologic bone remodeling around teeth or around dental implants is similar at the cellular level to bone remodeling that occurs in other bone tissues in the human body. Osteocytes, osteoclasts, and osteoblasts function to balance the processes of bone resorption and bone formation. The bone remodeling process is initiated by several factors, including parathyroid hormones (Shah et al., 2004), mechanical load (Yang et al., 2004), growth factors, and cytokines (Raggatt & Partridge, 2010), which result in the reorganization of the cellular and extracellular matrices (Ståhle-Bäckdahl et al., 1997) and changes in local vascularity (Matsuzaki, Wohl, Novack, Lynch, & Silva, 2007). This bone remodeling process leads to the synthesis and release of various neurotransmitters (Grässel, 2014), arachidonic acid (Dean et al., 2008), growth factors, metabolites, cytokines (Mundy, 1993), colony-stimulating factors (Raisz, 1999), and enzymes like cathepsin K (Crockett, Rogers, Coxon, Hocking, & Helfrich, 2011), matrix metalloproteinases (MMP) (Sasaki et al., 2007), and aspartate aminotransferase (Perinetti et al., 2003). During remodeling, osteoblasts mediate collagen degradation through the release of MMP-13, which results in the exposure of an arginyl-glycyl-aspartic acid motif for osteoclast binding (McHugh et al., 2000). Osteoblasts also release monocyte chemoattractant protein-1 (MCP-1) to recruit pre-osteoclasts. In addition, they express the receptor activator of nuclear factor kappa B ligand (RANKL), which then binds to its receptor, RANK, expressed on osteoclast precursor cells (Udagawa et al., 1999). The binding of RANK and RANKL directs the fate of hematopoietic progenitor cells to the osteoclast lineage and is a distinctive mark of osteoclast development. Indeed, conditional RANK (Dougall et al., 1999) or RANKL (Kong et al., 1999) knockout mice show no osteoclast differentiation. RANK-RANKL interaction is inhibited by the competitive binding of osteoprotegerin (OPG) to RANKL, which leads to the suppression of osteoclast differentiation (Wada, Nakashima, Hiroshi, & Penninger, 2006). Because of their important roles in osteoclastogenesis, RANK, RANKL, and OPG are tightly regulated during this process. Inflammatory bone remodeling through RANK/RANKL/OPG interaction is summarized in Fig. 1. In inflamed periodontal tissue, prostaglandin E2 (PGE2) and other inflammatory cytokines induce RANKL expression to promote osteoclastogenesis. PGE2 is the product of arachidonic acid conversion by cyclooxygenase-2 (COX-2). In periodontal tissue, Porphyromonas gingivalis lipopolysaccharide (PgLPS) stimulates PGE2 release (Noguchi et al., 1996) and increases the RANKL/ OPG ratio in primary human gingival fibroblasts (Belibasakis et al., 2007). The receptors for PGE2, E prostanoid (EP) 2 and EP4, play a role in PGE2-induced bone resorption (Minamizaki, Yoshiko, Kozai, Aubin, & Maeda, 2009). After PGE2 treatment, RANKL expression is increased in mouse calvarial osteoblasts. In addition to PGE2, the interleukin (IL)-6 cytokine may also play a role in osteoclastogenesis. IL-6 is produced by leukocytes, macrophages, periodontal ligament cells, and gingival fibroblasts in the periodontal tissue (Nebel, Arvidsson, Lillqvist, Holm, & Nilsson, 2013), and its levels is increased in response to IL-1, tumor necrosis factor a (TNFa), viruses, bacterial toxins, or lipopolysaccharide (LPS) (Morandini et al., 2010). Although IL-6 has both pro- and anti-inflammatory properties (Scheller, Chalaris, Schmidt-Arras, & Rose-John, 2011), it has been shown to increase RANKL and PGE2 expression in mouse calvarial osteoblasts (Palmqvist, Persson, Conaway, & Lerner, 2002) and in fibroblast-like synoviocytes of rheumatoid arthritis patients (Hashizume, Hayakawa, & Mihara, 2008) through the activation of the JAK/STAT signalling pathway. Co-stimulation of IL-6 and PGE2 leads to higher expression of COX-2, EP2, and EP4 and to enhanced

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R.A. Irwandi, A. Vacharaksa / Archives of Oral Biology 72 (2016) 66–74

Fig. 1. The interaction of RANK/RANKL/OPG in inflamed periodontal tissue. Periodontal pathogens stimulate the inflammatory cytokine release, including TNF-a, interleukin (IL)-1b, IL-6, and prostaglandin E2 (PGE2), in association with periodontitis. The inflammatory cytokines enhance the differentiation of osteoclast precursors and induce RANKL expression in gingival fibroblast, periodontal ligament cells and osteoblasts to promote osteoclastogenesis. Osteoblasts express the receptor activator of nuclear factor kappa B ligand (RANKL), which then binds to its receptor, RANK, expressed on osteoclast precursor cells. While osteoprotegerin (OPG) can competitively bind to RANKL and suppress osteoclast differentiation, the interaction of RANK and RANKL directs the fate of hematopoietic progenitor cells to the osteoclast lineage and is a distinctive mark of osteoclast development.

bone resorption in mouse calvarial osteoblasts (Liu, Kirschenbaum, Yao, & Levine, 2006). The pro-inflammatory cytokines, TNF-a (Singh, Gupta, Bey, & Khan, 2014) and IL-1b (Sánchez, Miozza, Delgado, & Busch, 2013), are also highly associated with the initiation of periodontitis. IL-1b enhances the differentiation of osteoclast precursors and induces RANKL expression in several cell types by promoting the interactions between interleukin (IL)-1, IL-1 receptor type 1 (IL-1R1), and IL-1 receptor accessory protein (IL-1RAcp), which activate the ERK and p38 MAPK signalling pathways (Steeve, Marc, Sandrine, Dominique, & Yannick, 2004; Mine, Makihira, Yamaguchi, Tanaka, & Nikawa, 2014). The activation of these pathways results in the increase of RANKL expression in primary human periodontal ligament cells (Kanzaki, Chiba, Shimizu, & Mitani, 2002; Nukaga et al., 2004), osteoblasts (Choi, Moon, Cha, Kim, & Yoo, 2005), gingival fibroblasts, and periodontal ligament fibroblasts (Belibasakis et al., 2007; Nukaga et al., 2004). TNFa also enhances RANKL-induced osteoclastogenesis via the coupling of TNFR1 and RANK signalling pathways (Zhang, Heulsmann, Tondravi, Mukherjee, & Abu-Amer, 2001). Pathogens, as well as endotoxins and related substances, can stimulate TNFa release from monocytes, macrophages, or T cells (Kitaura et al., 2013). Interestingly, recombinant RANKL also increases TNFa expression in mouse bone marrow (Zou, Hakim, Tschoep, Endres, & Bar-Shavit, 2001), where TNFa then stimulates the differentiation of macrophages into osteoclasts, as demonstrated by TRAP-positive staining. This process has been shown to be mediated by TNFa receptor type 1 (TNFR1) and type 2 (TNFR2) in vitro (Kobayashi et al., 2000) and in vivo (Lam et al., 2000). 3. The role of miRNA in bone remodeling In bone metabolism, some miRNAs are involved as stimulatory or inhibitory factors of osteogenesis. miRNA-194 stimulates osteoblast differentiation by targeting chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) in vitro (Jeong,

Kang, Hwang, Kim, & Koh, 2014). In mouse mesenchymal stem cells cultured in osteogenic medium, miR-194 is upregulated, while COUP-TFII is suppressed, by high levels of runt-related transcription factor 2 (RUNX2) (Lee et al., 2012). This result is reversed by the addition of anti-miR-194 (Jeong et al., 2014). miR-542-3p, on the other hand, inhibits osteoblast differentiation and proliferation by targeting BMP-7 and decreasing RUNX2-, type 1 collagen-, osterix-, and osteocalcin-specific mRNAs in mouse calvarial osteoblasts. miR-542-3p also inhibits bone formation and decreases the rate of mineral apposition in wild type and ovariectomized Balb/c mice (Kureel et al., 2014). MiRNAs also play a role in osteoclastogenesis. Protein inhibitor of activated STAT 3 (PIAS3) is a negative regulator for osteoclastogenesis by inhibiting transcriptional activity of microphthalmiaassociated transcription factor (MITF) (Kim et al., 2007; Hikata et al., 2009) and miR-9718 targets PIAS3 during osteoclast differentiation (Liu et al., 2014). Transfection of pre-miR-9718 into RAW 267.7 cells leads to lower expression of PIAS3, but higher expression of osteoclastogenic markers such as nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), c-Fos, MITF, and nuclear factor kappa B (NFkB). A higher number of TRAP-positive cells after pre-miR-9718 transfection also indicates an increase in osteoclastogenesis. Conversely, intravenous injection of antagomir-9718 in both wild-type and ovariectomized mice leads to restored bone mineral density (Liu et al., 2014). In contrast to miR-9718, miR-34a plays a role in the inhibition of osteoclastogenesis. In an in vivo model, miR-34a has been shown to target TGIF2 (Krzeszinski et al., 2014), which leads to the repression of TGFb-responsive genes (Melhuish, Gallo, & Wotton, 2001) and the inhibition of osteoclast differentiation (Quinn et al., 2001). miR-34a knockout mice exhibit decreased TRAP mRNA and TRAP-positive cell number, but increased bone quantity, when compared to wild type mice (Krzeszinski et al., 2014). miR-34a is significantly downregulated after an increase in RANKL or after treatment with rosiglitazone, a drug to induce bone loss. Further analysis shows that transfection of synthetic pre-miR-34a mimics into human

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peripheral mononuclear cells (PBMC) led to decreased TRAPspecific mRNA and TRAP-positive cell number. In contrast, transfection of an inhibitor of pre-miR-34a resulted in increased TRAP-specific mRNA and TRAP-positive cell number indicating miR-34a suppressed osteoclastogenesis in vitro (Krzeszinski et al., 2014). Delivery of miR-34a using chitosan nanoparticles to the ovariectomized and two-cancer-cell-cardiac-injection mouse models results in higher bone quantity and lower bone metastatic rate compared with control ovariectomized mice (Krzeszinski et al., 2014). Several miRNAs have been reported to be involved in the regulation of RANKL expression in various cell types, as summarized in Table 1. In cancer research, miR-335 has been shown to inhibit small cell lung cancer (SCLC) bone metastases by targeting insulin-like growth factor-1 receptor (IGF1R) and RANKL (Gong et al., 2014). Xenografts of the SCLC SBC-5, but not SBC-3, cell line triggers the formation of osteolytic bone lesions in non-obese diabetic/severe-combined and immune-deficient (NOD/SCID) IL2Rg null mice that lack mature T cells, B cells, and functional NK cells (Liu et al., 2014). A microarray study using the mParaflo1Microfluidic Biochip Microarray shows that 14 out of 833 miRNAs are downregulated in SBC-5 mice when compared with SBC-3 mice. This phenotype is thought to arise from the lack of miR-335 in SBC-5. Consistent with this, SBC-5 mice transfected with miR-355 show lower levels of both IGF1R and RANKL protein expression and lower incidence of bone lesions (Gong et al., 2014). The level of RANKL expression appears to be associated with osteoclastogenesis. In giant cell tumour of bone, miR-106b has been shown to inhibit osteoclastogenesis and osteolysis by directly targeting RANKL. Transfection of miR-106b into both giant cell tumour stromal cells and MG63 osteosarcoma cells directly targets RANKL mRNA, as shown by luciferase reporter assay, and reduces osteoclastogenesis in vivo (Wang et al., 2015). In contrast, glucocorticoid treatment induces secondary osteoporosis with bone loss and fragility fracture (Fraser & Adachi, 2009). In MC3T3E1 cells and an in vivo model, glucocorticoid treatment increases the level of RANKL while decreasing the level of miR-17/20a (Shi

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et al., 2014). The bone loss associated with glucocorticoid treatment seems to be partly rescued by the addition of miR-17/ 20a (Shi et al., 2014). These observations point to the role of miRNAs in RANKL regulation, which in turn affects bone metabolism. MiRNAs play a role in inflammatory mediator-induced osteoclastogenesis by targeting genes that encode inflammatory mediators or by ultimately affecting the release of these mediators and we summarized the relevant studies of it in Table 2. Rheumatoid arthritis shows chronic inflammatory characteristics similar to periodontitis (Agnihotri & Gaur, 2015). The presence of TNFa and IL-1b in rheumatoid arthritis can induce miR-146a expression in synovial fibroblasts (Nakasa et al., 2008). The NFkBdependent induction of miR-146a targets TRAF6 and IRAK1 (Taganov, Boldin, Chang, & Baltimore, 2006), which leads to the inhibition of TNFa, IL-1b, and IL-6 in human gingival fibroblasts (Xie et al., 2013). IL10-induced miR-187, on the other hand, decreases IL-6 production in primary human monocytes in vitro (Rossato et al., 2012). The consequences of miRNA modulation that affects inflammatory mediator release can be inducer-dependent and cell type-dependent. For instance, bleomycin-induced senescent HCA2 cells express higher miR-146a compared with untreated cells, resulting in reduced IL-6 and IL-8 production through IRAK1 inhibition (Bhaumik et al., 2009). PgLPS-induced miR-146 expression in human primary gingival fibroblasts, on the other hand, suppresses not only IL-6 but also IL-1b and TNFa via IRAK1 inhibition (Xie et al., 2013). Moreover, miRNA can also be induced by cytokines affecting other cytokine release. For example, IL-10induced miR-187, which suppressed TNFa and IL-6 through targeting TNFa-specific and NFkB inhibitor zeta-specific mRNA, respectively in human primary monocytes (Rossato et al., 2012). The effects of constitutive miRNA expression can vary between cell types. Fibroblast-like synoviocytes from rheumatoid arthritis patients express miR-146a, which reduces LPS-induced TNFa production by destabilizing TNFa-specific mRNA (Semaan et al., 2011). In SJL mice-derived bone marrow stem cells, on the other hand, miR-146a reduces PGE2 production by targeting PGE2

Table 1 The miRNAs thatare involved in bone metabolism and RANKL expression. miRNAs

Functions

Cell/tissue types

Reference

miR-194

Stimulation of osteogenesis through inhibition of COUP-TFII, RUNX2 suppressor protein.

McHugh et al. (2000)

miR-5423p

Inhibition of osteogenesis through suppression of bone morphogenetic protein (BMP-7)

miR-9718

Stimulation of osteoclastogenesis through inhibition of PIAS3

miR-34a

Inhibition of osteoclastogenesis through suppression of transforming growth factor ß-induced factor II (TGIFII)

miR-335

Inhibition of small cell lung cancer bone metastases by suppression of IGF1R and RANKL expression

miR-17 miR20a

Inhibition of glucocorticoid-induced osteoclastogenesis by suppressing RANKL expression

miR-106b

Inhibition of osteoclastogenesis and osteolysis through RANKL repression

primary mouse bone marrow stromal cells MC3T3-E1 cells In vitro Mouse calvarial osteoblasts In vitro Balb/c mice In vivo C57BL/6-derived bone marrow cells In vitro Wild type and ovariectomized C57BL/6 mice In vivo C57BL/6J-derived bone marrow cells In vitro miR-34a transgenic C57BL/6J mice In vivo SBC-5: small cell lung cancer cell line In vitro C57BL/6J mice In vivo Mouse calvarial osteoblasts: primary cells, MC3T3-E1 cell line, calvarial bone In vitro C57BL/6J mice In vivo Giant cell tumour In vitro Wild type and ovariectomized C57BL/6 mice In vivo

Miller et al. (2016)

Moffatt and Lamont (2011)

Morandini et al. (2010)

Murr (2009)

Nebel et al. (2013)

Nakasa et al. (2008)

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Table 2 The role of miRNAs in the release of inflammatory mediator inducing RANKL expression. miRNAs

Functions

Inducer

Cell/tissue type

Reference

miR-146

Suppression of IL-6 & IL-8 by targeting IRAK1

Senescent induction P. gingivalis LPS –

Human foreskin fibroblasts cell line, HCA2 In vitro Human primary gingival fibroblasts In vitro Human primary rheumatoid arthritis fibroblast-like synoviocytes In vitro Human primary monocytes In vitro Human primary chondrocytes In vitro Human primary umbilical monocyte In vitro SJL mice-derived bone marrow stem cells In vitro

Palmqvist et al. (2002) Ogata et al. (2014) Parikh et al. (2014)

Suppression of IL-6, IL-1b, and TNFa by targeting IRAK1 miR-346

Suppression of LPS-activated TNFa production by tristetrapolin stabilization of TNFa-specific mRNA

miR-187

IL-10

miR-199

Suppression of TNFa by targeting TNFa-specific mRNA Suppression of IL-6 by targeting NFkB inhibitory zeta Suppression of PGE2 synthesis by targeting COX-2 mRNA

miR-125b

Suppression of constitutive or LPS-stimulated TNFa production

–

miR-146a

Inhibition of PGE2 synthesis by targeting PGE2 synthase 2 mRNA

–

synthase 2-specific mRNA (Matysiak et al., 2013). In human primary chondrocyte, miR-199 also reduces PGE2 production by targeting COX-2-specific mRNA (Akhtar & Haqqi, 2012). However, constitutive miRNA expression can also modulate cytokine release constitutively and inductively, such as when miR-125b suppresses both constitutive and LPS-induced TNFa production in human primary umbilical monocytes (Huang et al., 2012). 4. MicroRNA dynamics in periodontal tissue and periodontitisrelated mechanisms The role of miRNAs in periodontal disease remains to be elucidated. Recent studies have investigated the profile of miRNAs in inflamed gingiva in comparison with healthy tissue summarized in Table 3. Two studies using the miRNA PCR array (SABioscience) reveal that several miRNAs are increased in inflamed periodontal tissue, including miR-181b, miR-19b, miR-23a, miR-30a, miR-let7a, and miR-301a, miR-128, miR-34a, and miR-381 (Lee et al., 2011; Na et al., 2016). Some miRNAs, including miR-211, miR-372, miR-656, are underexpressed in inflamed gingival tissue compared with healthy gingiva (Na et al., 2016). A study using the miRCURYTM array reports a greater than 2-fold increase in 91 miRNAs, and a greater than 2-fold decrease in 34 miRNAs in inflamed gingival tissue. Twelve of the 91 up-regulated miRNAs, including miR-1265p, miR-20a, miR-142-3p, miR-19a, let-7f, miR-203, miR-17, miR-223, miR-146a, miR-146b, miR-155, and miR-205, are related to inflammation, as predicted by TargetScan and the database on miRNA.org (Xie, Shu, Jiang, Liu, & Zhang, 2011) that contains miRNA target prediction based on miRANDA algorithm (Betel, Wilson, Gabow, Marks, & Sander, 2008). Another study using the Agilent Human miRNA Microarray shows that miR-150, miR-223, and miR-200b have the highest miRNA expression, while miR-199a5p and miR-214 had the lowest miRNA expression in inflamed tissue (Ogata et al., 2014). Interestingly, miR-150, miR-223, and miR-200b are predicted by Ingenuity Pathway Analysis (IPA) to associate with inflammatory disease, tissue injury, leukocytic and hormonal abnormalities, urological disease, and cancer (Ogata et al., 2014). A large-scale genome-wide microarray study (Stoecklin-Wasmer et al., 2012) that assessed over 1000 miRNAs in 198 gingival tissues shows that the expression of 159 miRNAs are significantly different when compared between healthy and inflamed gingiva. Four miRNAs, including miR-451, miR-223, miR-486-5p, and miR-3917, are significantly overexpressed, whereas seven miRNAs, including miR-1246, miR-1260, miR-141, miR1260b, miR-203, miR-210, and miR-205-3p, are under-expressed in inflamed gingiva. The five studies discussed above reveal distinct miRNA profiles for inflamed gingival tissues. This may be due to age and gender

–

Ouhara et al. (2014) Perinetti et al. (2003) Perri et al. (2012) Pasquinelli (2012)

variations among the study samples, differences in criteria for gingival inflammation, variable environmental exposure of individuals, especially at the tissue collection site, and differences in probes used for the microarrays. The inclusion of complex cells from the gingival tissue samples may also have contributed to the variability of miRNA expression patterns. Interestingly, however, three microarray studies consistently report the up-regulation of miR-223 in inflamed gingiva. miR-223 targets nuclear factor 1-A (NF1-A), and down-regulation of miR-223 by antisense transfection into RAW264.7 cells or by lentivirus-mediated silencing in a mouse model (Lee et al., 2011) has been found to reduce osteoclast formation by up-regulating NF1-A. PU.1, a transcription factor that defines osteoclast function, has also been found to enhance osteoclast formation by up-regulating miR-223 (Sugatani & Hruska, 2009). These studies suggest that miR-223 levels in inflamed gingival tissue may play a role in alveolar bone loss, which is a hallmark of periodontitis. The role of miRNAs in host immune response and inflammation through the regulation of inflammatory cytokines has been proposed in recent studies. Interestingly, based on miRNA profiling of overweight patients with periodontitis, obesity is thought to be a risk factor in periodontal tissue inflammation (Perri, Nares, Zhang, Barros, & Offenbacher, 2012). Inflammation-related miRNAs, including miR-18a and miR-30e, have been shown to be increased in obese patients, compared with non-obese patients, with healthy periodontium. While only miR-30e and miR-106b are significantly overexpressed in non-obese patient with periodontitis, miR-15a, miR-18a, miR-22, miR-30d, miR-30e, miR-103, miR106b, miR-130a, miR-142-3p, miR-185, and miR-210 are significantly overexpressed in obese patients with periodontitis. Functional analyses of target mRNAs using miRNA bioinformatic analysis and Gene Ontology (GO) shows that the miRNAs identified may be involved in the regulatory pathways of immunity and inflammation and the metabolism of collagen, lipid, and carbohydrate (Perri et al., 2012). A more recent study (Kalea et al., 2015) also reports the association of miR-200b with obesity and periodontitis, and its targeted genes are involved in wound healing and angiogenesis. Because of the small sample size of these studies, the role of miRNAs in regulating periodontitis and the impact of obesity in disease pathogenesis remain inconclusive. However, some miRNAs with increased expression in the group of obese patients with periodontitis may be significant. Among these upregulated miRNAs, miR-106b has been linked to carcinogenesis, cell-cycle regulation, inflammation, and bone metabolism. The detection of some miRNAs as a biomarker for periodontitis is also discussed. The use of saliva or gingival crevicular fluid as a diagnostic medium for detection of miRNA biomarkers is noninvasive and accessible (Xie et al., 2015). From the miRNAs that

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Table 3 Summary of recent reports in the profile of miRNA in inflamed gingiva compared with healthy gingival tissue. Author

Lee et al., 2011

Xie et al., 2012

StoecklinWasmer et al., 2012

Ogata et al., 2014

Na et al., 2016

Sample

Array

miRNAs

Criteria

Number

Age/ Gender

Inflamed gingiva: Chronic periodontitis PD > 5 mm; AL > 3 mm; Bone loss on radiograph Healthy gingiva: Low scores on BoP (under 10%); No site with PD > 3 mm No site presenting AL Inflamed gingiva: GI > 1; PD  5 mm; AL  3 mm at least 5 sites; Bone loss on radiograph Healthy gingiva: PD > 3 mm; AL < 1 mm; No bone loss on radiograph Inflamed gingiva: PD > 4 mm; AL  3 mm; BoP Healthy gingiva: PD  4 mm; AL  2 mm.; no BoP Inflamed gingiva: PD  6 mm AL > 6 mm Healthy gingiva: Edentulous residual ridges Inflammed gingiva: PD > 5 mm; AL > 3 mm; no bone loss on radiograph Healthy gingiva: Low scores of BoP (under 10%); no site with PD > 3 mm; no AL

N/A

N/A

10 periodontitis patients

miRCURYTM array microarray kit, v.11.0 (Exiqon, Denmark) containing 28 to 63 yr. 1,769 miRNA capture probes 4 males 6 females

10 healthy patients

21 to 51 yr. 4 males 6 females 13 to 76 Agilent Platform (Agilent Technologies, Santa Clara, CA, USA)with the miR-451 yr. probes containing 1,205 human and 144 human viral miRNAs. miR-223 miR-486-5p miR-3917

86 periodontitis patients

3 chronic periodontitis patients

Upregulated Downregulated RT2 miRNA PCR array system (SABiosciences, Frederick, MD, USA) containing 93 inflammatory miRNA primer

let-7a let-7c miR-130a miR-301a miR-520d miR-548a

N/A

miR-126 miR-20a miR-142-3p miR-19a let-7f miR-203 miR-17 miR-223 miR-146b miR-146a

miR-155 miR-205

miR-1246 miR-1260 miR-141, miR-1260b miR-203 miR-210 miR-205

N/A

human miRNA microarray 8  15k kit (Agilent Technologies, Santa Clara, CA, USA)

miR-150 miR-223 miR-200b

miR-144 miR-379 miR-222

N/A

RT2 miRNA PCR array system (SABiosciences, Frederick, MD, USA) containing 93 inflammatory miRNA primer

miR-128 miR-34a miR-381

miR-211 miR-372 miR-656

3 edentulous residual ridges N/A

GI = gingival index; PD = probing depth; AL = attachment loss; bleeding on probing = BoP.

relate to periodontal disease, miR-146aexpression in gingival tissue shows positive correlation to other clinical criteria for chronic periodontitis including probing depth and attachment loss (Motedayyen, Ghotloo, Saffari, Sattari, & Amid, 2015). miR-207, miR-495, and miR-376b-3p are proposed as a serum biomarker in the periodontitis rat model (Tomofuji et al., 2016), but its relevance to human disease requires further investigation. The inflammation of periodontal tissue frequently results in destruction of alveolar bone and connective tissue. The microRNAs potentially have a role as a regulator in host-pathogen responses and tissue homeostasis. Many studies have reported the function of

some miRNAs in periodontitis-related inflammation. Associating with gingival epithelial cell functions, miR-105, miR-203, miR-584, and miR-128 are shown to play an important role in the modulation of innate immune response against oral pathogens. miR-105 promotes IL-6 and TNFa production by targeting toll like receptor2 (TLR2) in human primary gingival keratinocytes, when cells are stimulated with heat-inactivated P. gingivalis (Benakanakere et al., 2009). In addition, P. gingivalis induces miR-203 expression in gingival epithelial cells, which leads to STAT3 activation through the inhibition of suppressor of cytokine signaling 2 (Moffatt & Lamont, 2011). Another miRNA, miR-584, is also induced by P.

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gingivalis LPS resulted in IL-8 upregulation in gingival epithelial cell line by repressing lactoferrin receptor (Ouhara et al., 2014). In human periodontal ligament cells (hPDLs), level of some miRNAs is shifted in response to P. gingivalis LPS stimulation shown by the Affimetrix array (Du et al., 2016). Subsequent validation using quantitative PCR shows that miR-21-5p, miR-498, and miR-548a-5p are significantly upregulated while miR-495-3p, miR-539-5p, and miR-7a-2-3p are downregulated. Another study in hPDLs shows that miR-146 targets IL-1 receptor-associated kinase 1 (Xie et al., 2013), thereby modulating pro-inflammatory secretion of cytokines, such as IL-1b and blocking the toll-like receptor signaling pathway after P. gingivalis LPS stimulation (Jiang et al., 2015). These results suggest a role of miRNAs in the inflammatory response to P. gingivalis infection. Recent evidence has indicated the regulatory functions of miRNAs in hPDLs involving in bone remodeling. The expression of some miRNAs is necessary for osteogenic differentiation of hPDLs. An increase of miR-132 by fluid shear stress (Qi & Zhang, 2014), or exogenous miR-146a (Hung et al., 2010), can stimulate hPDL cell proliferation and osteogenic differentiation demonstrated by alkaline phosphatase activity and in vitro mineralization. Expression of miR-17 is required in periodontal stem cells to suppress the ubiquitin regulatory factor one and promote the osteogenic differentiation of hPDLs (Liu et al., 2011). On the contrary, overexpressed miR-138 after interleukin (IL)-6 and LPS treatment reduces osteogenic differentiation in periodontal stem cells (Zhou et al., 2016), by targeting osteocalcin (OC) and Runt-related transcription factor 2 (RUNX2). The potential mRNA targets of miRNAs that have been identified in miRNA profiling studies have also been predicted. Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis have been used to define sets of genes in functional categories based on DAVID Bioinformatics Resources (Huang, Sherman, & Lempicki, 2009a; Huang, Sherman, & Lempicki, 2009b). From these analyses, 60 enriched miRNA sets have been identified, with target genes involved in immune/inflammatory responses and tissue homeostasis (Stoecklin-Wasmer et al., 2012). Interestingly, one target gene may be regulated by several miRNAs, for example, miR-218 is also shown to be able to target RUNX2 and decreases RUNX2 expression in undifferentiated human stem cells (Gay et al., 2014). Therefore, the mechanism by miRNAs appears to be tightly regulated. Although the function of miRNAs has been addressed, further studies are required to understand the miRNAs regulatory network in bone remodeling. 5. Conclusions MiRNAs regulate gene expression at the post-transcriptional level and affect numerous biological cascades. Bone metabolism, in particular, involves several miRNAs that ultimately influence cell interaction and signaling. Bone resorption may occur as a result of periodontal inflammation. RANKL expression in periodontal tissue can be induced by inflammatory mediators and leads to imbalanced bone metabolism. In periodontal resident cells, such as gingival fibroblasts and periodontal ligament cells, RANKL and inflammatory mediators can be modulated by miRNAs. In miRNA profiling studies of tissues isolated from individuals with periodontal disease, miR-223 has been consistently identified as a potential candidate miRNA to be further investigated in periodontitis-related processes. Although these studies point to an important role of miRNA-mediated epigenetic changes in tissue inflammation and alveolar bone loss, further investigation is still required to determine the function of miRNAs in the complex processes of periodontal tissue homeostasis and pathogenesis. Knowledge gained from future studies will be beneficial in

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