Archives of Oral Biology 73 (2017) 302–310

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Archives of Oral Biology

journal homepage: www.elsevier.com/locate/aob

Review

MicroRNA control of tooth formation and eruption

Ying Jina , Chenglin Wangb , Si Chengc , Zhihe Zhao, Prof.a,* , Juan Lia,*
a
Department of Orthodontics, State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, West China School of Stomatology,
Sichuan University, Chengdu, Sichuan, China
b
State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, Sichuan, China
c
Department of Orthopaedics, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China

A R T I C L E I N F O A B S T R A C T

Article history:
Received 20 January 2016 Tooth development involves epithelium invagination, mesenchyme aggregation, and epithelium–
Received in revised form 20 August 2016 mesenchyme communication. A sophisticated signaling pathway network regulates the differentiation
Accepted 22 August 2016 and crosstalk of multiple cell types in tooth germs and coordinates the broad spectrum of complex
processes. MicroRNAs (miRNAs), a class of small non-coding RNA species that have been relatively well
Keywords: studied over the last few years, are now proposed as important regulators of tooth developmental
MicroRNA signaling pathways as they repress cellular protein levels to provide a posttranscriptional gene
Tooth development regulation. In this review, we summarize the current knowledge of miRNA characteristics in regulating
Amelogenesis
morphogenesis, amelogenesis, dentin formation, and tooth eruption and how they interplay with the
Odontoblast
signaling molecules during these processes.
Tooth eruption
ã 2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
2. MiRNAs control morphogenesis and amelogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
2.1. TGF-b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
2.2. FGFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
2.3. WNT pathway . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
2.4. PITX2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
2.5. SOX2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
2.6. TBX1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
2.7. Membrane proteins . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
3. MiRNAs control dentin formation . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
3.1. DSPP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
3.2. NOTCH pathway . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
3.3. WNT pathway . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
3.4. BMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
3.5. RUNX2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
3.6. OSX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
3.7. KLF4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
3.8. DLX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
4. MiRNAs regulate tooth eruption . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309

Abbreviations: RISC, RNA-induced silencing complex; miRNAs, microRNAs; UTR, untranslated region; cKO, conditional knockout; laCL, labial cervical loop; liCL, lingual
cervical loop; DFC(s), dental follicle cell(s).
* Corresponding author at: Department of Orthodontics, State Key Laboratory of Oral Diseases, West China Stomatology Hospital, Sichuan University, #14, 3rd Section of
Renmin South Road, Chengdu 610041, China.
E-mail addresses: [email protected] (Z. Zhao), [email protected] (J. Li).

http://dx.doi.org/10.1016/j.archoralbio.2016.08.026
0003-9969/ã 2016 Elsevier Ltd. All rights reserved.
 Y. Jin et al. / Archives of Oral Biology 73 (2017) 302–310 303

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309

1. Introduction Biogenesis of miRNAs occurs by RNA polymerase II or III,

synthesizing what would become the hairpin loop of the primary
A tooth is formed from the tooth germ, an aggregation of cells, miRNAs(pri-miRNAs). The ribonuclease III enzyme Drosha then
which is organized into three parts: the enamel organ, the dental cleaves the pri-miRNAs to release stem-loop precursors miRNAs
papilla, and the dental sac or follicle. Tooth germ development (pre-miRNAs), which are exported from the nucleus to the
initiates from epithelial cells proliferating into the ectomesen- cytoplasm. Following translocation of the pre-miRNAs into the
chyme of the jaw, and a series of reciprocally inductive signaling cytoplasm, the pre-miRNAs undergo final cleavage by a second
pathways between the dental epithelium and ectomesenchyme RNA III enzyme, Dicer. Eventually, the mature miRNAs assemble
culminate with the formation of the enamel, dentin, dental pulp, into an RNA-induced silencing complex (RISC), which can impair
and periodontium—the supporting structures of a tooth. protein translation and/or modulates mRNA stability by pairing
Epithelial–mesenchymal interactions are under hormonal regula- with the 3’ untranslated region (UTR) of mRNAs (Foshay &
tion and are tightly coupled by several key-signaling pathways, Gallicano, 2007; He & Hannon, 2004). Because of imperfect base
including sonic hedgehog (SHH), bone morphogenetic protein pairing of miRNA seed sequences that represent a core of 5–7
(BMP), fibroblast growth factor (FGF), WNT, and NOTCH (Mitsiadis, nucleotides within the miRNA, one miRNA can target hundreds of
Lardelli, Lendahl, & Thesleff, 1995; Thesleff, 2003). Transcription mRNAs and can act selectively in different tissues, whereas one
factors such as PITX2, SOX2, Osterix, and RUNX2 govern the mRNA can have multiple binding sites for different miRNAs (He &
expression of proteins of these signaling pathways and play Hannon, 2004). Thus, miRNAs act as potent molecular managers
important roles during tooth development (Fig. 1). that can control several gene regulatory networks simultaneously.
MicroRNAs (miRNA) are the class of 19–25-nt non-coding MiRNAs contribute to each stage of tooth development from
single-stranded RNAs that negatively regulate gene expression morphogenesis, amelogenesis, and dentin formation to tooth
posttranscriptionally. Synthesis of miRNAs initiates in the nucleus. eruption by regulating growth, differentiation, and functional

Fig. 1. Schematic summary of dental epithelium and mesenchyme interaction during tooth development. Selected associated transcriptional factors and signaling pathways
involved in odontogenesis and mouse incisor regeneration are arranged in gray boxes. Black arrow indicates promotion, and red arrow indicates inhibition. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.)
304 Y. Jin et al. / Archives of Oral Biology 73 (2017) 302–310

activity of cells that constitute the tooth tissue. Understanding the inner dental epithelium gives rise to pre-ameloblasts and secreting
contribution of miRNAs to tooth formation requires cell-type ameloblasts, which eventually initiate enamel formation (Li, Yu, &
specific disruption of miRNA biogenesis. Conditional knockout Tian, 2013; Suryadeva & Khan, 2015; Ten Cate, 1996). However,
(cKO) of Dicer1 in the dental epithelium of mouse results in mouse incisors undergo a quite different development process. The
significant aberrations in tooth shape and enamel formation, but second enamel knots do not develop in the mouse incisor tooth
an increase in proliferation in the epithelium (Michon, Tummers, germs, but a special structure called cervical loops where the stem
Kyyronen, Frilander, & Thesleff, 2010). In addition, mutant mouse cells are cumulated develop at the proximal end of the incisor
with a cKO of Dicer showed an arrest or absence of teeth tooth germs. Rodent incisors grow throughout adult life, but are
development (Cao et al., 2010; Michon et al., 2010; Oommen et al., prevented from becoming excessively long by constant abrasion,
2012). Microarray and in situ hybridization analysis identified which is facilitated by the absence of enamel on one side of the
several miRNAs that showed differential expression profiles incisor. Labial cervical loop (laCL) can give rise to ameloblasts and
between dental epithelium and mesenchyme at different devel- eventually enamel, whereas lingual cervical loop (liCL) does not
opment stages (Michon et al., 2010; Oommen et al., 2012); a normally give rise to ameloblasts and enamel (Klein et al., 2008).
recently published study used miniature pigs as large-animal Inactivating Dicer1 in dental epithelium results in significant
models and reported differentially expressed miRNA expression aberrations in tooth shape and enamel formation. For example, in
during tooth morphogenesis in the early developmental stages of the Pitx2-Cre/Dicer1 cKO mice incisors, the expression of
the tooth germ (Li, Li, Song, Wang, & Liu, 2015). This review ameloblast differentiation markers dramatically decreased, in-
describes evolving biological concepts for the roles of miRNAs in cluding amelogenin and ameloblastin (Cao et al., 2010). In addition,
amelogenesis, dentin formation, and tooth eruption. the deletion of Dicer-1 function in the epithelium of developing
molars resulted in mild but significant malformations of tooth
2. MiRNAs control morphogenesis and amelogenesis crown shape (Michon et al., 2010). MiRNAs display considerable
enhancing or inhibitory functions in tooth morphogenesis and
Morphogenesis is a process initiating from epithelium invagi- epithelial cell differentiation in the development of both molars
nation to tooth bud initiation, generation, and shape. At the bud and incisors by targeting associated signaling pathways and
stage in developing dental epithelium, there forms a transient transcription factors (Fig. 2) (Table 1).
signaling center, termed “enamel knot.” The tooth germ then
undergoes the cap stage and the bell stage. At the bell stage in 2.1. TGF-b
developing molar teeth, several secondary enamel knots are
formed, which determine the locations and morphology of molar TGF-b superfamily ligands include BMPs, GDFs, anti-Müllerian
cusps. The secondary enamel knots express largely the same array hormone, Activin, Nodal, and TGF-bs. The TGF-b signaling
of multiple growth factors as the enamel knot. Subsequently, the pathway is involved in many cellular processes in tooth

Fig. 2. Enhancing or inhibiting effects of miRNAs on amelogenesis. miRNAs influence amelogenesis including ameloblast differentiation and morphogenesis by targeting
transcriptional factors and signaling pathway proteins.
 Y. Jin et al. / Archives of Oral Biology 73 (2017) 302–310 305

Table 1
miRNAs in tooth development.

Location MicroRNAs Potential target Sample source Origin Function Ref.

Dental epithelium miR-214 TGF-b1/Clusterin Molar tooth germ newborn mice (Khan et al., 2013;
Sehic et al., 2011)

miR200a-3p b-catenin/Pitx2 LS-8 cell line mice (Sharp et al., 2014)

miR-203 Bmper LS-8 cell line mice (Cao et al., 2013)
miR-200c/141 Noggin LS-8 cell line mice (Cao et al., 2013)
miR-720 Fgf8 P2 incisor mice (Juuri et al., 2012)
miR-200b Sox2 P2 incisor mice (Juuri et al., 2012)
miR-135a Bmpr-Ia and Bmpr-Ib Molar tooth germ/E14 mice (Kim et al., 2014)
miR-153 Lamp1 LS-8 cell line mice
miR-31 Tfrc LS-8 cell line mice
miR- 224 Slc4a4 and Cftr hOEs human (Fan et al., 2015)

miR96-5p Tbx1 LS-8 cell line mice (Gao et al., 2015)

Dental papilla miR-32,885-5p,586 Dspp hDPPC/impacted third molars human (Huang et al., 2011)
miR-34a, Bmp7/Hes1/Notch1,2 hFDPC/bell stage tooth buds human (Sun et al., 2014

miR-145 Osx/Klf4 mDPC/E18.5 molars mice (Liu et al., 2013)

miR-338-3p Runx2 mDPC6T mice (Sun et al., 2013)
miR-665 Kat6a/Dlx3 mDPC-23/OD-21/M06-G3 mice (Heair et al., 2015)
miR-27,663 Apc mDPC-23 mice (Kim et al., 2014;
Park et al., 2014)
Dental follicle miR-146a Egfr hDFC human (Chen et al., 2014)

LS-8 cell line, murine ameloblast-derived cells; hOEs, human fetal oral buccal mucosal epithelial cells; hDPPC, human dental pulp cells; hFDPC, human fetal dental papilla
cells; mDPC, mouse dental papilla cells; hDFC, human dental follicle cells; OD-21, rat odontoblast-like cells; M06-G3, mouse odontoblast-like cells. Red and down arrow
indicates inhibition effect, and blue and up arrows indicates promotion effect. (For interpretation of the references to colour in the table, the reader is referred to the web
version of this article.)

development. In particular, BMPs induced from the epithelium pronounced. In addition, as microarray analysis showed that the
play an important part in early tooth development and the transfection of anti-miR-214 led to markedly decreased expression
regulation of early tooth morphogenesis and differentiation of clusterin (a novel modulator of TGF-b signaling) and TGF-b 1 in
(Thesleff, 2003). In the BMP signaling, functional receptors in the treated molars, the possible targets of miR-214 could be
the target tissues are indispensable, including three type I BMP clusterin and TGF-b1, which are two important proteins during
receptors: BMPR-IA, BMPR-IB, and ActR-I; and two type II BMP amelogenesis process and located close to each other in the dental
receptors: BMPR-II and ActR-II (Miyazono, Kamiya, & Morikawa, epithelium of murine molar tooth germs (D'Souza, Happonen,
2010). Inactivation of Bmpr-Ia resulted in the arrest of tooth Ritter, & Butler, 1990; French et al., 1993; Jevnaker & Osmundsen,
development at an early stage (Li et al., 2011). 2008; Khan et al., 2013; Lee et al., 2008).
To date, studies have identified several miRNAs regulating
amelogenesis by targeting TGF-b family members. MiR-135a shows 2.2. FGFs
an important role in regulating enamel formation by targeting
BMPR-IA and BMPR-IB (Kim et al., 2014). BMPR-1A and BMPR-IB The FGF family currently consists of 23 structurally related
protein expression was downregulated by miR-135a overexpression polypeptides that share a similar internal core and have a
in molar tooth germs after kidney capsule transplantation. Further- characteristically high binding affinity for both heparin and FGF
more, the translational level of pSMAD-1,5,8, downstream of BMP receptors (FGFRs). FGFs have been implicated in regulating dental
signaling, also decreased after the overexpression of miR-135a. On epithelial stem cells. FGF3 stimulates the proliferation of inner
the contrary, miR-135a expression was abrogated by the exogenous enamel epithelial cells; FGF7 promotes the proliferation of outer
expression of BMP2 delivered by BMP2-soaked protein beads, which enamel epithelial cells; FGF10 maintains dental epithelial stem cells;
were implanted adjacent to tooth germs. MiR-200c/141 contributes and FGF8 as an autocrine regulator also maintains the epithelial stem
to epithelial cell differentiation through a mechanism related to cells in incisors (Harada & Ohshima, 2004; Harada et al., 2002; Juuri
upregulation of E-cadherin amelogenin and downregulation of et al., 2012). Among these FGFs, Fgf8 30 UTR is linked to miR-720 as
noggin, an antagonist of BMP signaling (Cao et al., 2013). Evidence revealed by luciferase assay. In addition, because the posttranscrip-
indicates that miR-200c directly repressed noggin expression by tional regulation of Fgf8 by miRNAs is in line with the loss of
binding on LS-8 murine dental epithelium-like cells. In addition, epithelial homeostasis observed in the Shh-Cre; Dicer-1 cKO, the
miR-203 targeted and repressed BMP antagonist Bmper through a fine-tuning of Fgf8 might be necessary to maintain the homeostasis
conserved sequence in its 3’-UTR to further regulate cytodifferenti- of dental epithelium in the incisor (Juuri et al., 2012).
ation of dental epithelium-like cells (Cao et al., 2013). In vivo studies
showed that miR-214 indirectly affected amelogenesis by targeting 2.3. WNT pathway
TGF-b pathways (Khan, Sehic, Khuu, Risnes, & Osmundsen, 2013;
Sehic, Risnes, Khuu, Khan, & Osmundsen, 2011). In the anti-miR- The WNT signaling pathway is one of several key conserved
214-treated molars, the enamel exhibited evidence of hypominer- intercellular signaling pathways in animals and is dynamically active
alization; the enamel was porous with remnants of organic in tooth-forming regions at all stages of tooth development (Liu &
material, and the surface roughness after etching was also less Millar, 2010). It is suggested that WNT signaling plays a positive role
306 Y. Jin et al. / Archives of Oral Biology 73 (2017) 302–310

in dental initiation (Liu et al., 2008), and b-catenin is an upstream palatine. In particular, TBX1 is expressed in the epithelium of
activator of enamel knot formation (Jarvinen et al., 2006). In addition, developing tooth germs during the early stage. Evidence indicates
WNT signaling also regulates tooth morphology initiation at the bell that TBX1 regulates the timing of ameloblast differentiation,
stage (Sarkar & Sharpe, 2000). Conversely, at the secretory stage, epithelial cell proliferation, and the subsequent production of
WNT pathway inhibition shows a positive role in enamel formation enamel proteins (Gao et al., 2015). Further, the dose of miR–96 s
(Millar et al., 2003). With regard to the fine-tuning of WNT signaling, fine-tunes the TBX1 expression and hence regulates cell prolifera-
Sharp et al. found that miR-200a-3p directly targeted the b-catenin tion versus differentiation. Recently, researchers found that the
30 UTR and repressed b-catenin expression in cultured LS-8 cells Tbx1 30 UTR contains a highly conserved miR-96 binding element,
(Sharp et al., 2014). After being transducted with pLL-miR-200a and miR-96-Tbx1 functions in a regulatory loop to maintain the
lentivirus, the LS-8 cells showed increased E-cadherin expression correct levels of TBX1 and to regulate the proliferation versus
and decreased cyclin D2 and Lef-1 expression, and the morphology differentiation of dental progenitor cells. In the laCL, where the
of LS-8 cells changed into clusters of cells that more closely miR-96 expression is low and TBX1 is high, TBX1 maintains the low
resemble epithelial cells (Sharp et al., 2014). levels of miR-96, which is important for TBX1 regulation of dental
stem cell niche. However, in ameloblasts, the increased expression
2.4. PITX2 of miR-96 is one of the modulators that downregulate the TBX1
expression to facilitate ameloblast differentiation (Gao et al., 2015).
Pitx2 also known as pituitary homeobox 2 gene has proven to be
the gene mutated in Rieger Syndrome type I (RGS I) that includes 2.7. Membrane proteins
tooth abnormalities as one of its primary features. Evidence
suggests that PITX2 may control dental progenitor cell prolifera- In addition to these signaling molecules and transcription
tion and differentiation, as a positive regulator of FGF8 and a factors, some important membrane proteins are also regulated by
repressor of BMP4 signaling (Lu, Pressman, Dyer, Johnson, & miRNAs during amelogenesis. MiR-153 and MiR-31, two potential
Martin, 1999). Recently, researchers have revealed several post- regulators, have their predicted target mRNAs, lysosomal-associ-
transcriptional regulatory pathways of PITX2 in the dental ated membrane protein 1 and transferrin receptor, respectively,
epithelium. PITX2 expression caused an upregulation of miR- which are considered to be the ameloblast membrane-bound
200c/141by binding to the miR-200c/141 promoter, and miR-200c/ receptors for EMP debris (Bartlett et al., 2006; Lacruz et al., 2013;
141 knockout mice showed defects in enamel formation (Cao et al., Shapiro et al., 2007). In addition, miR-224 was reported to bind to
2013). PITX2 activates miR-200a-3p, another cluster of the miR- the 30 UTRs of Slc4a4 and cystic fibrosis transmembrane conduc-
200 family. Meanwhile, miR-200a-3p reciprocally represses PITX2 tance regulator mRNAs, which are highly expressed during
and b-catenin expression (Sharp et al., 2014). Interestingly, the ameloblast differentiation as ion transporters to maintain pH
combination of PITX2 and miR-200a-3p reprograms mesenchyme homeostasis and support enamel mineralization (Fan et al., 2015).
cells to anamelogenin-expressing dental epithelial cells, which
involved an upregulation of the stem cell marker SOX2, E-cadherin, 3. MiRNAs control dentin formation
and ameloblast-specific factors and decreased expression of
mesenchyme markers (Sharp et al., 2014). In addition, endogenous Dentin formation is another important process other than
PITX2 also activates miR-203, which targets BMP antagonist Bmper enamel formation in tooth development. From the beginning of
to further regulate BMP signaling (Cao et al., 2013). The miR-96- tooth formation, mesenchyme condenses beneath the dental
Tbx1 regulatory pathway may also indirectly negatively regulate epithelium. The signaling from the oral ectoderm, epithelium bud,
PITX2 expression and transcriptional activity and therefore fine- and enamel knot induces mesenchymal condensation, followed by
tune amelogenesis activated by PITX2 (Gao et al., 2015). cytodifferentiation and eventually dentin formation (Arana-
Chavez & Massa, 2004; Liu et al., 2008. Condensed mesenchymal
2.5. SOX2 cells commence dental papilla formation. The dental papilla cells
located near the epithelium differentiate into preodontoblasts. The
SOX2, one of the SRY-related HMG box transcription factors, preodontoblasts continue to differentiate into polarized odonto-
maintains the pluripotency of early embryonic cell and is equally a blasts, secretory odontoblast, and terminally differentiated odon-
co-inducer of iPS cell (Takahashi & Yamanaka, 2006). Ablation of toblasts lying in the outermost layer of the dental pulp (Balic,
SOX2+ cells in mice results in a disruption of epithelial tissue Aguila, & Mina, 2010) (Fig. 1). During this process, dentin
homeostasis (Arnold et al., 2011). It is reported that SOX2 is a extracellular matrix proteins, including both collagen and non-
specific marker for the dental epithelial stem cells and is regulated collagen proteins, are essential for mineralized dentin formation.
by the tooth initiation marker FGF8. Besides, luciferase assay Dentin sialophosphoprotein (DSPP) as one of the noncollagen
reveals that the miR-200b activity is linked to Sox2 30 UTR by 37.6% proteins has been implicated as having a regulatory function in
compared to scramble miRNA. Meanwhile, in situ hybridization in odontoblast differentiation (Balic et al., 2010; Qin, D’Souza, & Feng,
P2 mouse incisor revealed the miR-200b expression pattern 2007). The signaling molecules of BMP, FGF and WNT families and
matched with the ectopic SOX2 expression observed in Shh-Cre; transcription factors such as RUNX2 are also involved in
Dicer-1fl/fl mutant, and involved instellate reticulum, preamelo- odontoblast differentiation. Thus, miRNAs that target these
blasts, and ameloblasts on the labial side and the liCL. In other proteins would be regarded important for dentin formation
words, the SOX2 expression in dental epithelial stem cells is (Fig. 3) (Table 1).
possibly regulated by miR-200b posttranscriptionally (Juuri et al.,
2012). 3.1. DSPP

2.6. TBX1 Dspp transcription is highly specific for odontoblast lineage

during the secretory stage of tooth development (Begue-Kirn,
T-box genes encode a large group of transcription factors Krebsbach, Bartlett, & Butler, 1998; Chen, Gluhak-Heinrich, Wang,
characterized by a 180-amino-acid DNA-binding domain. Tran- Wu, & Chuang, 2009). Dentin sialoprotein (DSP) and dentin
scripts of Tbx1 are localized to the epithelium of the early facial phosphoprotein (DPP) are two major noncollagenous dentin
processes, including fronto-nasal, medial and lateral nasal, and matrix proteins. Both of them are encoded by the Dspp gene
 Y. Jin et al. / Archives of Oral Biology 73 (2017) 302–310 307

Fig. 3. Schematic of miRNA regulation of odontoblast differentiation and dentin formation. miRNAs control odontogenesis by targeting transcriptional factors, signaling
pathway proteins, and epigenetic factors.

and are localized within the dentin matrix. Evidence has been level were downregulated 72 h after miR-34a mimicked transfec-
accumulating that DSP and DPP are essential for dentin biominer- tion, but were rescued by anti-miR-34a transfection. Thus, the
alization and/or the formation of the structural diversity of tooth authors proposed that miR-34a suppresses NOTCH1, resulting in
layers, including mantle dentin, predentin, and orthodentin. The cell differentiation and upregulation of DSPP in developing dental
teeth of Dspp knockout mice display a widened predentin zone and mesenchyme. Another study suggested that miR-34a directly
develop defective dentin mineralization, similar to human represses NOTCH signaling by targeting Notch2 and Hes1, a direct
dentinogenesis imperfecta type III (Sreenath et al., 2003). Dual downstream target gene of NOTCH signaling, and indirectly
luciferase reporter assays and qRT-PCR identified that the DSPP upregulates differentiation genes such as Dspp. The authors
expression of dental pulp cells is regulated posttranscriptionally by utilized a dual luciferase assay to find that miR-34a directly binds
miR-32, miR-885-5p, and miR-586 during odontoblast differentia- to the 30 UTR of Notch2 and Hes1, thereby posttranscriptionally
tion, although the complex regulatory network between miRNA regulating mRNA levels. On the contrary, miR-34a was significantly
and mRNAs remains to be determined (Huang et al., 2011). upregulated after JAG1 (a ligand of the Notch pathway) treatment
Interestingly, a recent study using RISC immune precipitation and and downregulated by DAPT (an inhibitor of the Notch pathway)
biotin-labeled miR-665 pull-down study showed that miR-665 treatment. Thereafter, miR-34a and NOTCH signaling functioned in
may contribute to the alteration in the chromatin status of Dspp a regulatory loop to fine-tune the odontogenic differentiation in
and Dmp1 by targeting Kat6a expression, which controls the stem cells (Sun et al., 2014).
acetylation of histone H3 and H4 tails and the alteration in MOZ/
MORF complex composition (Heair et al., 2015). 3.3. WNT pathway

3.2. NOTCH pathway WNT families and its members also show a positive expression
in dental mesenchyme and play important roles in odontogenesis.
NOTCH signaling is a highly conserved cell signaling system. It is Mesenchyme-specific inactivation of b-catenin revealed a critical
commonly accepted that NOTCH is critical for keeping stem cells at role of Wnt/b-catenin signaling in the activation of the mesen-
an uncommitted/naive state (Koch, Lehal, & Radtke, 2013). NOTCH chymal odontogenic potential during early tooth development
receptors and their ligands are expressed in both dental epithelial (Chen, Lan, Baek, Gao, & Jiang, 2009). It is also suggested that Wnt/
and mesenchymal cells (Mitsiadis, Romeas, Lendahl, Sharpe, & b-catenin signaling is functionally significant for root odonto-
Farges, 2003). Accumulating evidence indicates that NOTCH genesis in postnatal mouse. Recent studies using MDPC-23
signaling plays a critical role in tooth morphogenesis and odontoblastic cell revealed two miRNAs that regulate odontoblas-
odontoblastic differentiation as well as in tooth repair and tic differentiation by targeting Wnt/b-catenin signaling pathways.
regeneration (Mitsiadis, Henrique, Thesleff, & Lendahl, 1997; The mRNA of the adenomatous polyposis coli (Apc) gene, which is
Mitsiadis et al., 1995). Study utilizing dental papilla cells revealed associated with the Wnt/b-catenin signaling pathway, has both
that transfection with miR-34a mimics enhanced DSPP expression, miR-663 and miR-27 binding sites in its 3’UTR region. APC
but decreased ALP levels. Furthermore, NOTCH1 and BMP7 protein expression was suppressed significantly by miR-663 and miR-27,
308 Y. Jin et al. / Archives of Oral Biology 73 (2017) 302–310

and this downregulation of APC expression triggered the activation 3.7. KLF4
of Wnt/b-catenin signaling through the accumulation of b-catenin
in the nucleus, eventually promoting the differentiation of MDPC- Kruppel-like factor 4 (KLF4) has been extensively investigated
23 cells to odontoblasts (Kim, Park, Lee, Park, & Kim, 2014; Park for its roles in cell proliferation and cytodifferentiation. Studies
et al., 2014). demonstrated that its positive expression in polarizing odonto-
blasts was associated with the increased expression of odontoblast
differentiation marker genes such as Dmp and Dspp (Feinberg et al.,
3.4. BMPs
2007; Lin et al., 2011). A feedback loop relating to KLF4 expression
and odontogenesis regulation has been discovered in dental papilla
BMPs belong to the TGF-b superfamily. Accumulated evidence
mesenchymal cells. Firstly, the miR-145 inhibits not only OSX but
indicates that BMPs play pivotal roles in the regulation of
also KLF4 expression by directly binding to the 30 UTR of their
embryonic development and pattern formation. Apart from its
mRNA. Secondly, KLF4 on the contrary directly represses miR-143
important role in epithelial differentiation as we described above,
transcription, which seemed to be the upstream of miR-145. Thus,
the BMP signaling pathway is vital for odontogenesis (D’Souza
it inhibited the odontoblast differentiation in part through miR-
et al., 1990). To date, studies have shown that miRNAs can regulate
145 (Liu et al., 2013).
odontogenesis by modulating the BMP signaling pathway. In
cultured dental papilla cells, the BMP7 protein level was down-
3.8. DLX
regulated after miR-34a mimicked transfection, while it was
upregulated after miR-34a inhibited transfection. The author
The homeodomain gene Dlx3 is recognized as another critical
proposed that by targeting Bmp7, miR-34a might indirectly inhibit
regulator of craniofacial and postnatal skeletal development
the expression of ALP, an osteogenic marker gene, and regulate the
(Duverger et al., 2012; Hyun & Kim, 2009; Isaac et al., 2014).
cytodifferentiation of human dental papilla cells toward odonto-
Mutations in Dlx3 in humans have been associated with tricho-
genesis (Wan et al., 2012).
dento-osseous (TDO) syndrome, an autosomal dominant disorder
characterized by abnormalities in the thickness and density of
3.5. RUNX2 bones and teeth (Choi et al., 2010; Dong et al., 2005; Hyun & Kim,
2009). It is found that Dspp is the direct target of DLX3 in
RUNX2, a member of the Drosophila runt family, is a key odontoblasts (Duverger et al., 2012). Recently, it is reported that
transcription factor associated with osteoblasts and odontoblast miR-665 shows a low expression compared to the high expression
differentiation (D’Souza et al., 1999; Miyazaki et al., 2008). RUNX2 of DLX3 during differentiation of mouse odontoblast and human
is indicated to inhibit the terminal differentiation of odontoblasts. dental pulp cells. Furthermore, miR-665 directly targeted Dlx3
In Runx2 transgenic mice, odontoblasts were poorly differentiated mRNA and decreased DLX3 expression and its downstream targets
and dentin formation was also abnormal (Miyazaki et al., 2008). (Runx2,Osx,Dspp) in mouse odontoblasts. Thus, miR-665 attenuates
RUNX2 expression is upregulated in the condensed dental odontoblast maturation by targeting Dlx3 mRNA (Heair et al.,
mesenchyme but downregulated in the differentiated odonto- 2015).
blasts during cytodifferentiation (Miyazaki et al., 2008). Thus, fine-
tuning of Runx2 by miRNA may play an important role during 4. MiRNAs regulate tooth eruption
odontoblast differentiation. Recently, a study showed that miR-
338-3p could regulate RUNX2 expression posttranscriptionally Tooth eruption is a complex process regulated by osteoblast and
during the odontoblast cytodifferentiation. By using MDPC6T cells, osteoclast interaction and affected by various factors. There are
the authors mocked the terminal differentiation of odontoblasts, two types of eruption processes, namely limited eruption (e.g.,
and they found that the expression level of odontoblast human dentition, rodent molars) and continuous eruption (e.g.,
differentiation marker genes such as Dspp and Dmp was rodent incisors), which are regulated by different molecules and
upregulated. Meanwhile, they found Runx2 is a direct target of mechanisms. We refer to limited eruption in this review.
miR-338-3p, as RUNX2 was downregulated after overexpression of It is accepted that tooth eruption requires alveolar bone
miR-338-3p, and the Runx2 30 UTR reporter was suppressed by resorption and formation regulated by the dental follicle (Cahill
mi38-3p (Sun et al., 2013). Thus, miR-338-3p can promote & Marks, 1980; Marks & Cahill, 1984). Dental follicle cells (DFCs),
odontoblast differentiation by directly suppressing RUNX2 originating from cranial neural crest mesenchyme, have potential
expression. of multidirectional differentiation (Pan et al., 2010). They can
differentiate into periodontal ligament, cementum, and proper
3.6. OSX alveolar bone. To study the mechanism of eruption, researchers
found an appropriate model, that is, the DFCs of an unerupted teeth
Osterix (OSX) is another osteoblast-specific transcription from a patient with cleidocranial dysplasia (CCD), an autosomal
factor expressed in dental mesenchymal cells. Its expression dominant hereditary disease characterized by impacted supernu-
pattern was similar to that of RUNX2 at an early stage of tooth merary teeth. For example, a study used samples from patients
development. But unlike RUNX2 expression, Osx transcripts with CCD to reveal a novel mutation of Runx2, which may
remained intense in odontoblasts and dental pulp cells at the late contribute to the abnormal function of DFCs, hindering tooth
stages (Chen et al., 2009). It is suggested that OSX could induce cell eruption. It is also suggested that RUNX2 may be regulated by miR-
differentiation and tooth-related gene expression at the later 146a, because when miR-146a was overexpressed or inhibited, the
stages of tooth development by upregulating Dspp and Dmp gene expression of RUNX2 was significantly altered (Chen et al., 2014)
expression (Liu et al., 2013). A posttranscriptionally fine-tuning of (Table 1).
Osx has been discovered in dental papilla mesenchymal cells. In addition to DFCs, the secretory ameloblasts also probably
The miR-145 inhibits OSX expression by directly binding to the participate in the process of tooth eruption, especially in the
30 UTR of its mRNA and therefore retarding odontoblast differenti- breakdown of the connective tissue (Ten Cate, 1996). Some factors
ation. In addition, the miR-143 inhibited OSX expression and that are secreted from ameloblasts stimulate macrophage chemo-
odontoblast differentiation through the miR-145 pathway (Liu taxis, which has been suggested to be important in tooth eruption.
et al., 2013). Among these factors, clusterin and TGF-b1 are two proteins that
 Y. Jin et al. / Archives of Oral Biology 73 (2017) 302–310 309

have been found to be associated with macrophage chemotaxis Choi, S. J., Song, I. S., Feng, J. Q., Gao, T., Haruyama, N., Gautam, P., . . . Hart, T. C.
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