Involvement of Noncoding RNAs in the Differentiation of Osteoclasts

As the most important bone-resorbing cells, osteoclasts play fundamental roles in bone remodeling and skeletal health. Much effort has been focused on identifying the regulators of osteoclast metabolism. Noncoding RNAs (ncRNAs) reportedly regulate osteoclast formation, differentiation, survival, and bone-resorbing activity to participate in bone physiology and pathology. The present review intends to provide a general framework for how ncRNAs and their targets regulate osteoclast differentiation and the important events of osteoclastogenesis they are involved in, including osteoclast precursor generation, early differentiation, mononuclear osteoclast fusion, and multinucleated osteoclast function and survival. This framework is beneficial for understanding bone biology and for identifying the potential biomarkers or therapeutic targets of bone diseases. The review also summarizes the results of in vivo experiments and classic experiment methods for osteoclast-related researches.

1. Introduction 1.1. A Brief Introduction to Osteoclasts. Multinucleated osteoclasts are closely related to orthodontic tooth movement, as they can resorb mineralized bone and, together with osteoblasts, initiate the bone remodeling cycle [1]. Osteoclasts are also essential for continuous tooth movement as they remove adjacent bone and the necrotic tissue generated by periodontal ligament hyalinization [2,3]. Moreover, bone is remodeled throughout life, which consists of osteoclasts resorbing old bone and osteoblasts synthesizing new bone. When the balance between these two processes is broken, many skeletal disorders occur [4,5]. Enhanced osteoclast formation and activity contribute to bone loss of osteoporosis, rheumatoid arthritis, and osteolysis of metastatic tumors [6][7][8].

A Brief
Introduction to Noncoding RNAs. Noncoding RNAs (ncRNAs) are defined as RNAs that do not encode proteins [12] and include "housekeeping" rRNAs, tRNAs, small nuclear RNAs, and small nucleolar RNAs (snoRNAs) [13]. But ncRNAs are far more than these. Studies have shown that there are only approximately 20,000 proteincoding genes in the human genome, while at least 90% of the genome is actively transcribed which implies that about 80% of the genome is functional noncoding genes [14]. Although the transcripts of some such genes are suspected to be transcriptional noise, ncRNAs are important biological determinants and are recognized as a breakthrough for disease treatment in mechanism research, drug development, and new biomaterial invention [13,15]. In physiology and development of life, they control gene expression in multiple levels, including chromatin architecture/epigenetic memory, transcription, RNA splicing, editing, translation, and turnover [12]. ncRNAs are subdivided into two major classes according to their size: small ncRNAs (<200 nt) and long ncRNAs (lncRNAs, >200 nt) [14]. Small ncRNAs consist of microRNAs (miRNAs) which regulate posttranslational gene silencing, piwi-interacting RNAs (piRNAs) which silence retrotransposable elements in the genome and regulate DNA methylation, snoRNAs which modify RNAs other than rRNAs, etc. [16][17][18]. lncRNAs, comprising long or large intergenic ncRNAs (lincRNAs), circular RNAs (circRNAs), etc., are involved in chromatin remodeling, transcriptional regulation, and posttranscriptional processing and thus play important roles in regulating gene expression during development, differentiation, and disease [16,19,20].
Since miRNA is the most widely studied type of ncRNAs, its naming is more orderly. miRNA names include sequential numerical identifiers to distinguish them from each other [21]. Lettered suffixes indicate that the miRNAs differ at only one or two positions in paralogous sequences-for instance, miR-29a, miR-29b, and miR-29c [21]. And "5p" or "3p" at the end of miRNA names indicate that they are processed from the 5 ′ and 3 ′ end arms-for example, miR-142-5p and miR-142-3p [21].

Literature Collection
The research was carried out in the MEDLINE database (PubMed research engine): ((((miRNA) OR lncRNA) OR ncRNA) AND ((osteoclast differentiation [tw]) OR (osteoclastogenesis))). This search retrieved 1577 articles. Then, considering that most of the ncRNA researches were carried out after 2008, we narrowed the publish date to January 1, 2008, to March 1, 2020. We also excluded reviews and added additional filter criteria of language, using the search strategy as follows: ((((((miRNA) OR lncRNA) OR ncRNA) AND ((osteoclast differentiation [tw]) OR osteoclastogenesis))) NOT "review"[Publication Type]) AND ("2008/01/01" [PDat]: "2020/03/01"[PDat]) AND English [lang]). This search retrieved 1352 articles. Afterwards, we manually assessed the titles and abstracts of the collected articles and excluded those not relevant, which means that the articles are not mainly focused on the relationship between

Involvement of ncRNAs in Osteoclast Differentiation
The factors that regulate osteoclastogenesis, such as ncRNAs, natural compounds, and epigenetic mechanisms, have been investigated [22]. In vitro experiment showed that the expression levels of 518 lncRNAs, 207 mRNAs, 37 miRNAs, and 24 circRNAs fluctuated during osteoclastogenesis of murine RAW264.7 cells [22], which indicated a role of ncRNAs in the regulation of osteoclastogenesis. For example, osteoclasts cannot be produced from bone marrow HSCs deficient in mature miRNAs [23][24][25][26]. Moreover, small interfering RNA-(siRNA-) mediated silencing of the Drosha enzyme complex (which promotes miRNA biosynthesis) or the RNA-induced silencing complex (RISC) (which mediates miRNA binding to its target messenger) blocks osteoclastogenesis [23,27]. Table 1 summarizes a list of osteoclast-related ncRNAs with validated targets and functions in regulating osteoclastogenesis. Table 2 summarizes a list of ncRNAs implicated in biological events in osteoclasts in in vivo studies.
In the following sections, we will discuss miRNAs and lncRNAs according to their functioning stages in osteoclastogenesis ( Figure 2). To our knowledge, ncRNAs and their targets are usually involved in more than one stage of osteoclastogenesis. To give a more accurate network of ncRNAs, we tend to classify the functioning stages of ncRNAs according to their mostly described functions.
3.1. Involvement of ncRNAs in OCP Generation. Osteoclasts are derived from HSCs, which give rise to CMPs, and the latter yield GMPs. GMPs further differentiate into macrophage/osteoclast/dendritic cell precursors, which are known as OCPs [9].
3.1.1. M-CSF, CSF1R, and PU.1. The differentiation from early-stage CSF1Rosteoclast progenitors to late-stage CSF1R + OCPs involves various differentiation, proliferation, and antiapoptotic signals. These signals are initiated by M-CSF produced by the neighboring cells of osteoclasts, including osteoblast and bone marrow stromal cells. M-CSF could induce the proliferation of OCPs, promote cell survival, and upregulate the expression of RANK [28]. In this process, circRNA_28313, miR-195a, and M-CSF together form a circRNA-miRNA-mRNA axis, in which circRNA_28313 relieves miR-195a-mediated suppression on M-CSF by acting as a competing endogenous RNA, therefore modulating osteoclastogenesis of bone marrow-derived macrophages (BMMs) [29].
Besides M-CSF, CSF1R and PU.1 also play key roles in the generation of OCPs. CSF1R could transmit M-CSF signals. And PU.1, which is a member of the Ets family, could regulate CSF1R transcription through Ets-binding sites on a CSF1R promoter. It was observed that deletion of CSF1R and PU.1 resulted in the lack of both macrophages and osteoclasts [30]. Overexpression of miR-155 suppresses PU.1 expression and activity, resulting in inhibited TRAP expression, osteoclast differentiation, and bone-resorbing activity, as well as repressed mononuclear osteoclast fusion and syncytia formation [31].
3.1.2. IL-15. IL-15 stimulates the differentiation of osteoclast progenitors into OCPs. The possible mechanism might be that IL-15 induces RANKL expression in osteoblasts and stromal cells, indirectly promoting osteoclastogenesis. Moreover, IL-15 is reported to exhibit a synergistic effect with RANKL in osteoclast differentiation by inducing activation of extracellular signal-regulated kinase (ERK), a specific subtype of mitogen-activated protein kinase (MAPK) [32]. As miR-212 negatively targets IL-15, the inhibition of miR-212 gives rise to upregulated IL-15 and thus promotes osteoclastogenesis [33].
3.1.3. IL-11. IL-11 is identified as a necessary factor that promotes osteoclast differentiation. The possible mechanism involves the downstream JAK1/signal transducer and activator of transcription-3 (STAT3) signaling pathway, in which activated STAT3 induces the expression of c-Myc, an important factor in osteoclastogenesis [34]. IL-11 also downregulates GM-CSF expression and sustains the pool of osteoclast progenitors [32,35]. During the process of osteoclastogenesis, GM-CSF reveals duration-relevant biphasic effects as it exhibits positive effect on osteoclast proliferation and formation in short-term treatment while it inhibits osteoclastogenesis in long-term treatment [36]. So IL-11 might work as an inhibitor of GM-CSF long-term effect and thus promotes osteoclast differentiation. miR-124 plays a negative role in cancer-induced osteoclast differentiation and activity through targeting IL-11 [37].

Involvement of ncRNAs in the Early Differentiation of
OCPs. During this stage of osteoclastogenesis, the change of OCPs is mainly manifested in the interaction and expression of internal molecules, and more osteoclast-specific molecules are expressed.
3.2.1. RANK/RANKL and Related Molecules. RANK and CSF1R are both transmembrane signaling receptors required for osteoclast differentiation and activation. RANKL and M-CSF bind to and activate RANK and CSF1R, respectively, which initiates the early stage of osteoclastogenesis [10]. Downregulation of miR-503 and miR-144-3p facilitates this stage of osteoclastogenesis by targeting RANK. Conversely, their overexpression inhibits osteoclastogenesis, demonstrated by reduced TRAP staining, decreased TRAP-positive cell numbers, and suppressed expression of osteoclast-specific genes [38,39]. Overexpression of lncRNA-NONMMUT037835.2 is also reported to inhibit osteoclastogenesis, as NON-MMUT037835.2 could downregulate RANK expression and the subsequent NF-κB/MAPK signaling pathway [40].

Negative
(1) Exhibits reduced osteoclast numbers and activity and inhibited osteoclast differentiation (2) Inhibits bone metastasis of breast cancer cells and cancer cell-induced osteolysis and shows relieved bone lesion [37,201] miR-146a CIA mice injected with miR-146a Negative Less osteoclasts at the border of the pannus and bone [56] miR-503 (1) Silencing of miR-503 with and antagomir (2) Overexpression of miR-503 with agomir Negative (1) Increases RANK protein expression and bone resorption and reduces bone mass (2) Inhibits bone resorption and bone loss [38] miR-145 Bilateral OVX-operated mice treated with a miR-145 agomir Negative Attenuates trabecular disconnection and separation and reverses the increase in the RANKL to OPG ratio [60] miR-17~92 miR-17~92 osteoclast conditional knockout mice Negative Exhibits decreased connectivity density, BV/TV, BMD, Tb.N, and Tb.Th and increased trabecular separation and bone resorption [155] 8 Stem Cells International as well as the protein level of RANK and RANKL, by targeting fibroblast growth factor 21 (FGF21) [50], which is able to alter the RANKL/OPG ratio and promote osteoclastogenesis by indirect effects through other cell types or secreted factors [51]. Ephrin type-A receptor 2 (EphA2) is suggested to be a miR-141 target in rhesus monkey osteoclasts [52]. Excessive expression of EphA2 in OCPs induces osteoclast differentiation. And EphA2 is involved in osteoblast-osteoclast interaction as the ratio of RANKL/OPG decreases in osteoblasts lacking EphA2. There is also some hypothesis that EphA2 may participate in osteoclast autoregulation between osteoclasts at different stages of differentiation [53].

NF-κB and Related
Molecules. NF-κB is a key regulator of osteoclastogenesis, of which the induction is a hallmark event in the cell fate determination of osteoclasts. In the canonical pathway of NF-κB activation, TAK1 phosphorylates and activates inhibitor of NF-κB kinase subunit β (IKKβ). IKKβ together with IKKα then phosphorylates and degrades inhibitor of NF-κB (IκB), resulting in an NF-κB dimer entering the nucleus [61].
Upregulation of miR-99b gives rise to enhanced NF-κB activity as it is reported to target insulin-like growth factor 1 receptor (IGF1R), whose signaling impedes NF-κB activity [33,67]. IGF1, the ligand of IGF1R, is required for normal osteoclast differentiation demonstrated by IGF1 knockout presenting a decreased osteoclast number [68]. IGF1 is also a possible target of miR-422a [47]. IGF2 is a peptide hormone that can modulate osteoclastogenesis through IGF1R activation and may participate in osteoblast-osteoclast interactions [69,70]. IGF2 is thought to be the likely target of miR-483-5p promoting osteoclast differentiation and inhibiting apoptosis as miR-483-5p directly inhibits the expression of IGF2 and IGF2 could in turn reverse the effects of miR-483-5p [71].

NFATc1 and Related
Molecules. NFATc1 is an essential transcription factor for osteoclastogenesis, which is inducted  Figure 2: Important regulators involved in ncRNAs regulating osteoclastogenesis. ncRNAs function through various regulators to participate in different stages of osteoclastogenesis, including OCP generation, early differentiation, mononuclear osteoclast fusion, and multinucleated osteoclast function and survival. The regulators are positioned by the stage of osteoclastogenesis they are mainly involved in. Positive regulators are listed above the cell figures, while negative regulators are listed below them. 10 Stem Cells International mainly through two ways-initial induction and autoamplification. In the initial induction, activated NF-κB is recruited to the NFATc1 promoter and cooperates with NFATc2 to activate the initial induction of NFATc1. The autoamplification refers to RANKL-RANK binding cooperating with immunoreceptors to activate the calcium signals, which induces NFATc1 autoamplification via calcineurin. Activator protein 1 (AP-1, containing c-Fos and c-Jun), which is recruited to the NFATc1 promoter concomitantly, is also critical for this autoamplification [79]. NFATc1 expression and activation are supported by the downregulation of miR-124 as it directly targets NFATc1 [80,81]. In the meantime, through the inhibition of miR-124, endothelial progenitor cell-(EPC-) derived exosomal lncRNA-MALAT1 induces migration and osteoclastogenesis of BMMs [82]. miR-1897 also targets NFATc1 and downregulates its expression. And lncRNA-MIRG functions as a molecular sponge to modulate the inhibitory effect of miR-1897 on NFATc1 and therefore enhances NFATc1 expression [83].
lncRNA-LINC00311 has been implicated in osteoclastogenesis by inducing osteoclast proliferation and differentiation while suppressing apoptosis via targeting delta-like 3 (DLL3), whose downregulation activates the Notch signaling pathway. In this way, overexpression of LINC00311 elevates the expression levels of neurogenic locus notch homolog protein 2 (Notch2) and TRAP [101]. Notch2 has been shown to interact with NF-κB, and both of them are recruited to the NFATc1 promoter and enhance the expression of NFATc1 [102,103]. The knockdown of lncRNA-DANCR could reduce the promotion effect of compression force on osteoclastogenesis via Jagged 1, a ligand of Notch2. DANCR is also reported to promote the expression of IL-6 and tumor necrosis factor-α (TNF-α), of which both have bone-resorbing activity in OCPs [104,105].

Other ncRNA Targets Involved in the Early
Differentiation of OCPs. NFATc1, cooperating with AP-1, PU.1, and microphthalmia-associated transcription factor (MITF), generates an osteoclast-specific transcriptional complex and induces the expression of various osteoclast-specific genes, including TRAP, CALCR, CTSK, and osteoclastassociated receptor (OSCAR) [79]. MITF is also thought to be important for the survival of OCPs as it is capable of regulating the antiapoptotic protein Bcl-2 expression in the osteoclast lineage [30]. Downregulation of miR-155, miR-340, miR-141, miR-219, and miR-133a promotes osteoclastogenesis by targeting MITF, and the overexpression of these miR-NAs results in decreased expression of osteoclast marker genes and repressed osteoclast differentiation and boneresorbing activity [31,55,121], although the positive effect of miR-133a on osteoclastogenesis is also reported [92]. LPS-induced miR-29b elevates osteoclast survival through targeting and decreasing Bcl-2-modifying factor (BMF), which can bind to Bcl-2 and induce the apoptosis of osteoclasts [122].
Sirtuin 1 (SIRT1) suppresses osteoclast differentiation by inhibiting ROS production as well as transient receptor potential cation channel subfamily V member 1 (TRPV1) activation. Upregulation of miR-506 supports osteoclastogenesis by targeting at 3 ′ -UTR of SIRT1, while a miR-506 inhibitor suppresses osteoclastogenesis demonstrated by TRAP staining, impaired functions, reduced the multinuclear cell number, and downregulated the expression of osteoclast markers and correlated genes [128].
The miR-29 family, including miR-29a, miR-29b, and miR-29c, is also involved in osteoclast lineage commitment as it negatively targets G protein-coupled receptor 85 (GPR85) and CD93, which are promoters of monocyte differentiation into macrophages, and thus interferes with osteoclastogenesis indirectly [129].

Involvement of ncRNAs in Mononuclear Osteoclast
Fusion. After the early differentiation, OCPs are committed to the osteoclast lineage and expressing various osteoclastspecific genes, including TRAP. These cells are also known as TRAP-positive mononuclear osteoclasts. These TRAPpositive mononuclear osteoclasts then migrate to each other, make contacts with neighboring cells by membrane protrusion, and subsequently fuse to become multinucleated osteoclasts [130].

DC-STAMP and Related
Molecules. Dendritic cellspecific transmembrane protein (DC-STAMP), a downstream gene of NFATc1, is essential for the cell-cell fusion of osteoclasts [30,131]. Downregulation of miR-7b and miR-30a boosts the fusion of TRAP-positive mononuclear osteoclasts by targeting DC-STAMP and modulating the expression of their downstream fusogenic and regulatory genes [132,133]. Inhibition of DC-STAMP by miR-7b also modulates the expression of NFATc1 and OSCAR via the immunoreceptor tyrosine-based activation motif-(ITAM-) immunoreceptor tyrosine-based inhibition motif (ITIM) network [132]. miR-30a overexpression in OCPs suppresses the DC-STAMP/c-Fos/NFATc1 signaling pathway and thus inhibits the formation of osteoclasts, actin ring, and resorbing pit [133]. The overexpression of miR-26a has the same effect as miR-30a, as it targets and downregulates connective tissue growth factor (CTGF), which induces DC-STAMP expression [134,135]. lncRNA-MAYA is involved in the regulation of DC-STAMP indirectly as it mediates the activation of yes-associated protein 1 (YAP1), which upregulates CTGF expression, leading to elevated cancer cellinduced osteoclastogenesis and bone resorption [136]. miR-222-3p is also reported to modulate the expression of DC-STAMP [137].

Rho GTPases.
The rho family of GTPases, including Ras homolog gene family member A (RhoA), Ras-related C3 botulinum toxin substrate 1 (Rac1), and cell division control protein 42 (CDC42), is involved in various osteoclastogenesis events, such as osteoclast commitment, proliferation, migration, fusion, and maturation [138]. Upregulation of miR-31 could control cytoskeleton organization and support mononuclear osteoclast fusion by targeting RhoA and negatively 12 Stem Cells International regulating RhoA expression at an appropriate level as both overexpression and suppression of RhoA show a negative effect on osteoclastogenesis [139]. miR-124 may inhibit the expression of RhoA and Rac1, leading to reduced mononuclear osteoclast migration [80]. Upregulation of miR-29 is implicated in cytoskeletal organization-involved cell migration by targeting CDC42 and SLIT-ROBO Rho GTPaseactivating protein 2 (SRGAP2), a negative regulator of Rac1 [129]. Downregulation of miR-17 is reported to increase the fusion of osteoclasts possibly via the miR-17-proteintyrosine phosphatase-(PTP-) EphA4 axis or directly targeting vav guanine nucleotide exchange factor 3 (Vav3), thus promoting ITGB3-dependent Vav3-mediated activation of Rac1/Rac2 [140]. 3.3.4. CD226. CD226 is involved in the process of mononuclear osteoclast fusion into multinucleated osteoclasts. It is reported that the formation of multinucleated osteoclasts is impaired when treated with the extracellular domain of CD226, while the formation of TRAP-positive mononuclear osteoclasts is not affected. The promoter of CD226 is regulated by AP-1, which mediates the inhibition effect of NFATc1 on CD226 promoter activity [142]. CD226 is one of the potential targets of miR-422a, and its expression was found to be negatively correlated, albeit not significantly, with that of miR-422a [47].

Involvement of ncRNAs in Multinucleated Osteoclast
Function and Survival. Osteoclasts are polarized when initiating bone resorption. Polarized osteoclasts form three distinct membrane domains: a ruffled border, a sealing zone, and a functional secretory domain. At the sealing zone, the actin cytoskeleton forms an attachment ring and anchors osteoclasts to the bone matrix [143,144]. The ruffled border inside the sealing zone consists of distinctive villous-like complexes of the plasma membrane, which harbor massive "spike-like" vacuolar proton pumps [143]. Via these proton pumps, the ruffled membrane transports protons into the resorption compartment to dissolve minerals, while osteoclasts secrete the lytic enzymes TRAP and pro-CTSK to degrade bone matrix proteins [5]. This erodes the underlying bone. The degradation products of collagen and other matrix components are processed in osteoclasts and transported through the cell and exocytosed through the functional secretory domain [144].
3.4.1. CTSK and CALCR. CTSK and CALCR are activated by a transcriptional complex containing NFATc1 and its cooperators including AP-1, p38MAPK, PU.1, and MITF [30,145]. CTSK is able to degrade type I collagen and regulate osteoclast actin ring formation and therefore influence bone resorption activity [146]. CALCR mediates the antiapoptotic effect of calcitonin on mature osteoclasts, leading to increased osteoclast survival while decreasing their resorption activity [129]. Downregulation of miR-141 and miR-190 improves osteoclastogenesis by targeting CALCR [55]. Upregulation of miR-29 increases osteoclast-mediated bone resorption by targeting CALCR [129]. However, it is also reported that miR-29b expression decreases during osteoclast differentiation in vitro, and its overexpression impairs TRAP expression, lacunae generation, and collagen degradation, which are indicators of osteoclast activity [147].

NFI-A. Reduced nuclear factor I/A (NFI-A) expression
is essential for the terminal differentiation of osteoclasts, as well as granulocytes and monocytes. Upregulation of miR-29 positively regulates osteoclastogenesis by targeting NFI-A [129]. NFI-A is also targeted by miR-223 [64]. The role of miR-223 in osteoclastogenesis is still controversial as both of positive and negative effects are mentioned [27,64,148,149]. The expression of miR-223, which is typically downregulated during osteoclastogenesis, is regulated by the competitive binding of NFI-A and CCAAT-enhancer-binding proteins α (C/EBPα) to their promoters. NFI-A maintains a low level of miR-223 expression, whereas C/EBPα upregulates miR-223 expression [150]. miR-223 is a component of the PU.1/miR-223/NFI-A/CSF1R positive feedback loop. In this loop, PU.1 expression is induced by M-CSF-stimulated production of pre-miR-223. And PU.1, together with C/EBPα, stimulates the expression of miR-223 reversely. miR-223 then downregulates NFI-A, leading to the upregulation of CSF1R, which actuates osteoclastogenesis, as well as the expression of PU.1, MITF, and other transcription factors [23,25,64], while in the condition that miR-223 expression is extremely low, its translational repression on NFI-A will be relieved, resulting in blocked osteoclastogenesis [64].

Cbl and c-Src.
Osteoclast adhesion and migration involve the tyrosine phosphorylation of casitas B lineage lymphoma (Cbl) and PI3K, as well as the activation of c-Src. Besides its role as an adaptor protein, Cbl is also a ubiquitin ligase which could target CSF1R and c-Src and regulate the activation of Rac [151,152]. Downregulated miR-9 and miR-181a promote Cbl expression and activity by targeting Cbl, which is also a potential target of miR-422a [47,153]. Downregulation of miR-222-3p supports osteoclast adhesion and migration by targeting c-Src, while pit formation activity of multinucleated osteoclasts is decreased by miR-222-3p gain-of-function [137]. CREB-binding protein (CBP, also known as phosphoprotein membrane anchor with glycosphingolipid microdomains 1 (PAG1)), which is also a potential target of miR-422a, inhibits c-Src activity in a dose-dependent manner. Moreover, introduction of CBP leads to suppressed formation of both actin rings and the ruffled border, resulting in decreased bone-resorbing activity [47,154]. Downregulation of miR-17 elevates c-Src activation by targeting PTP, which dephosphorylates the inhibitory pY527 of c-Src to activate c-Src signaling. miR-20 and miR-92, which are also members of the miR-17~92 cluster genes, also have potential target sites on PTP mRNA, indicating the 13 Stem Cells International possibility that they play similar negative regulation roles in osteoclastogenesis to that of miR-17 [140,155].
3.4.4. APC. miR-27a obviously suppresses osteoclast differentiation and activity as miR-27a overexpression markedly disrupts F-actin ring structure and decreases the pit formation of osteoclasts. Adenomatous polyposis coli (APC) is one of the targets of miR-27a, by which miR-27a enhances the inhibitory effect of estrogen on osteoclastogenesis and bone-resorbing activity [84]. APC can stabilize microtubules directly by interacting with microtubules via its microtubulebinding regions or indirectly by interacting with end-binding protein 1 (EB1). Inhibited APC activity gives rise to decreased microtubule stability in osteoclasts, leading to reduced sealing zone formation as well as decreased bone resorption activity. Phosphorylation by GSK-3β decreases the microtubule-binding ability of APC, while AKT enhances APC activity by phosphorylating GSK-3β and decreasing its activity [156].
3.4.5. MMP9. Matrix metalloproteinase 9 (MMP9), which is activated by RANKL stimulation, could promote OCP migration and accelerate osteoclastic resorption via increased degradation of collagen types I and IV [157]. Downregulation of miR-218 augments osteoclastogenesis by negatively targeting MMP9 [158]. MMP13 is also involved in osteoclast differentiation and activation as it is reported to activate pre-MMP9 and complement the activity of MMP9 in dissolving the bone matrix [159]. miR-126-5p targets MMP13 and represses its expression at a posttranscriptional level to regulate osteoclast differentiation and bone-resorbing activity in a giant cell tumor [160].
3.4.6. MMP14. MMP14 is crucial for osteoclast migration. As a type of secreted MMP, MMP14 is able to regulate osteoclast podosome function, and its collagenolytic activity is thought to be helpful for the osteoclast movement through a collagen fiber network. MMP14-deficient osteoclasts exhibit decreased bone resorption activity [161]. miR-133a targets MMP14 and thus represses osteoclast differentiation and bone-resorbing activity [55].
3.4.7. Fas and FasL. The Fas-Fas ligand (FasL) system, which provides an important apoptotic mechanism for osteoclasts, is activated by FasL binding to a Fas receptor [67,162]. miR-21 is involved in the inhibiting and proapoptotic effects of estrogen during osteoclastogenesis as estrogen downregulates miR-21 biogenesis. Decreased miR-21 expression increases the protein levels of FasL, a target of miR-21, and thus induces osteoclastic apoptosis [163]. miR-21 also participates in the regulation of osteoclast differentiation and recruitment in aging bones. Decreased miR-21 levels in aging bones result in osteocyte apoptosis and thus induce osteoclastogenesis by high-mobility group box-1-(HMGB1-) receptor for advanced glycation end product (RAGE) signaling [164]. HMGB1-RAGE signaling plays important roles in regulating actin cytoskeleton reorganization in osteoclasts, thereby participating in RANKL-induced osteoclastogenesis [165]. So miR-21 is also likely to be involved in the regulation of osteoclast bone-resorbing activity.
3.4.8. Rab27a. Rab27a is capable of regulating the transport of lysosome-related organelles and cell surface receptors and thus participating in the modulation of osteoclast bone-resorbing activity. Additionally, Rab27a-deficient osteoclasts display multinucleation, larger cell appearance, and abnormal actin ring formation [166]. miR-124 directly targets Rab27a. Overexpression of miR-124 reduces the protein level of Rab27a, thus inhibiting osteoclastogenesis and impairing the functions of osteoclasts [167].
3.4.9. Other ncRNAs and Their Targets Involved in Osteoclast Function and Survival. Interferon regulatory factor 1 (IRF1), one of the targets of miR-132, suppresses osteoclast maturation and/or activity under the mechanism related to proinflammatory cytokines like IL-12 and IL-18 [33,168].
Thrombospondin 1 (THBS1), a glycoprotein present in a variety of cell types, is one of the targets of miR-let-7e. THBS1 has been identified to be involved in osteoclast-mediated resorption under an undefined mechanism [33,169].
miR-16-5p suppresses RANKL-induced osteoclastogenesis in a giant cell tumor of bone and disrupts the formation of the F-actin ring, which is essential for osteoclast boneresorbing activity. However, the direct target of miR-16-5p functioning is undefined [170].

Involvement of ncRNAs with Undefined Targets and Functioning Stages
3.5.1. lncRNA-Bmncr. The expression of lncRNA-Bmncr gradually decreases during RANKL-induced osteoclastogenesis, which reaches the lowest level at 72 h. The overexpression of Bmncr reduces osteoclast numbers and bone-resorbing capacity and decreases the expression of osteoclast-specific genes, while knockdown of Bmncr has opposite effects [171].

Enhancer RNAs.
Enhancer RNAs (eRNAs) are a kind of lncRNA present in the 5′-flanking regions or intronic regions of genes which initiate bidirectional RNA biosynthesis and are involved in the differentiation of various cell types [173,174]. Deletion of the eRNA regions of neuropilin 2, DC-STAMP, and NFATc1 significantly inhibits the formation of TRAP-positive multinucleated cells, indicating that their expression positively regulates osteoclastogenesis. And eRNAs also functions by increasing the expression of RNAs encoding osteoclast-related proteins [173].

Discussion
Bone remodeling involves various biological events of osteoblasts and osteoclasts, all of which are regulated by a precise network of systemic and local factors, including ncRNAs 14 Stem Cells International [175,176]. When the network is unable to compensate for the difference between bone resorption and synthesis, unbalanced bone remodeling will cause diseases [177]. As a kind of ncRNAs with numerous numbers, miRNAs downregulate gene expression by either of two posttranscriptional ways: mRNA cleavage or translational repression. The former one occurs when the mRNA has sufficient complementarity to the miRNA; otherwise, the latter one occurs [178]. The way lncRNAs work is mainly through the interaction with miR-NAs as molecular sponges [179].
Most studies of the functions of ncRNAs in the regulation of osteoclast activity have focused on ncRNA expression levels, the effects of ncRNA mimics/inhibitors, and ncRNA target genes. Apart from osteoclast pathological experiments, RAW264.7 cells and BMMs have been the most frequently used sources of osteoclasts in such studies. RAW264.7 is a mouse monocyte macrophage leukemia cell line developed by injection of Abelson leukemia virus [180]. BMMs are produced by culturing nonadherent bone marrow cells from the femur and tibia in the presence of M-CSF [181]. The J774 and U-937 cell lines have also been used to generate osteoclasts [180]. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis is one of the most frequently used methods for analyzing the gene expression levels of ncRNAs and osteoclastspecific genes [58,97,101,180,182,183]. Microarray analysis is also widely used for detecting gene expression levels, as well as identifying novel regulators of osteoclastogenesis [141,184]. Microcomputed tomography enables the assessment of the structural parameters involved, such as bone volume per total volume (BV/TV), mean trabecular number (Tb.N), mean trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) [43,180,185]. Immunoblotting, as well as qualitative and semiquantitative analyses, is extensively used for the detection and characterization of proteins [186]. Chromatin immunoprecipitation (ChIP) enables the analysis of interactions between proteins and DNA in chromosomal fragments and the identification of the binding sites of transcription factors and other proteins [180,187]. Additionally, four web-based miRNA databases, miRBase (http://www.mirbase.org), TargetScan (http://www.targetscan. org), PicTar (http://www.pictar.mdc-berlin.de), and miRanda (http://www.microRNA.org), are essential for bioinformatics analysis [108]. Assays of pit formation, actin ring formation, cell migration, macrophage commitment, and apoptosis are also widely used to evaluate the functions and generation of osteoclasts [86,98,115,129,147,153].

Conclusion
With the profound research in bone physiology and diseases related to bone metabolism disorder, the essential role of ncRNAs in osteoclastogenesis is gradually recognized. In this review, we summarized the current knowledge of ncRNAs involved in important osteoclast differentiation events, including OCP generation, early differentiation, mononuclear osteoclast fusion, and multinucleated osteoclast function and survival. Nevertheless, there are still some unknown mechanisms for ncRNAs regulating osteoclast dif-ferentiation, and further work is needed to supplement and perfect the effects of ncRNAs, particularly those of lncRNAs, circRNAs, and eRNAs, on the functions of osteoclasts.

Conflicts of Interest
All authors have no conflicts of interest.

Authors' Contributions
Yi Zhao and Yunfei Zheng conceived and designed the study strategy. Lingfei Jia was responsible for the reference collection. Yi Zhao wrote the manuscript and prepared the tables and figures. Yunfei Zheng reviewed and edited the manuscript. Weiran Li and Yunfei Zheng were responsible for the study supervision. All authors reviewed the manuscript.