MicroRNAs as biomarkers for dental diseases

==Abstract==

MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators of diverse biological processes by mediating translational repression or mRNA degradation of target genes. Recent studies discovered miRNAs in saliva, and these miRNAs are promising candidates for use as biomarkers of dental diseases. In this review, the results of miRNA studies in the dental field are presented, and a brief overview of the current progress, limitations, and perspectives regarding miRNA biomarkers for dental diseases is given.

==Keywords==

microRNA ; Biomarker ; Saliva ; Periodontitis

==MicroRNAs==

MicroRNAs (miRNAs) are a class of small single-stranded noncoding RNAs that were first discovered in ''C. elegans''  and later shown to be evolutionarily conserved across many animal species, including humans  [[#bib1|[1]]] , [[#bib2|[2]]]  and [[#bib3|[3]]] . MicroRNAs, which are approximately 22 nucleotides in length, act as key regulators of diverse biological processes by mediating translational repression or mRNA degradation of their target genes  [[#bib4|[4]]]  and [[#bib5|[5]]] . The mode of action of these regulators is through imperfect complementary binding to the 3′ untranslated region (3′ UTR) of target mRNAs  [[#bib4|[4]]]  and [[#bib6|[6]]] . Typically, a single miRNA has the potential to simultaneously control the translation of hundreds of genes  [[#bib5|[5]]]  and [[#bib7|[7]]] . To date, more than 2500 genes encoding miRNAs have been identified in the human genome  [[#bib8|[8]]]  and [[#bib9|[9]]] .      

The biogenesis of miRNAs begins with the production of mRNA-like polyadenylated primary transcripts (pri-miRNA) by RNA polymerase II. Subsequent miRNA maturation requires two RNase III proteins, Drosha and Dicer. These two proteins may collaborate in the stepwise processing of miRNAs, and also have key roles in the process of miRNA-mediated gene regulation. The mature single-stranded miRNAs are eventually incorporated into a ribonucleoprotein complex, the RNA-induced silencing complex (RISC). RISC acts to downregulate gene expression via mRNA cleavage or translation repression: the miRNA component acts as a guide [[#bib10|[10]]] , [[#bib11|[11]]] , [[#bib12|[12]]] , [[#bib13|[13]]]  and [[#bib14|[14]]] .      

==Circulating microRNAs==

Serum and plasma contain large amounts of stable miRNAs, and these therefore have potential to serve as biomarkers for specific physiological and pathological conditions such as cancer, transplant rejection, cardiac injury, infection, and others [[#bib3|[3]]] , [[#bib15|[15]]] , [[#bib16|[16]]] , [[#bib17|[17]]]  and [[#bib18|[18]]] . The salivary transcriptome, which contains more than 3000 RNA species, including miRNAs, was recently released, and salivary miRNA biomarkers are emerging as tools for the detection of oral cancer and systemic diseases [[#bib19|[19]]] , [[#bib20|[20]]] , [[#bib21|[21]]] , [[#bib22|[22]]]  and [[#bib23|[23]]] . The miRNAs in saliva have advantages for biomarkers compared with other salivary biomarkers like proteins, mRNAs, DNAs and bacterial products. Even aside from the distinctive function of miRNA as post-transcriptional regulator, miRNAs are stably present in saliva and the similarity between miRNA profiles of saliva and other body fluids provide high availability as biomarkers for various human diseases [[#bib21|[21]]] , [[#bib24|[24]]]  and [[#bib25|[25]]] .      

Saliva sampling is a non-invasive, cheap, easy-to-access alternative to traditional tissue and blood sampling [[#bib26|[26]]]  and [[#bib27|[27]]] . Saliva is an accurate indicator of bodily conditions, and salivary biomarkers may serve as early diagnostic tools. Saliva diagnostics is considered to be a highly promising alternative to classic environmental epidemiology [[#bib28|[28]]]  and [[#bib29|[29]]] . The ease of sampling and cost effectiveness of saliva-based tests provide advantages over traditional techniques for large scale population-based screening studies and also in situations where repeated sampling is required, such as for monitoring and managing disease progression [[#bib30|[30]]] . Lab-on-a-chip (LOC) technologies are beginning to be used in clinical settings for point-of-care (POC) diagnostics [[#bib31|[31]]] . Saliva samples have been used successfully for POC detection of multiple disease entities, including some that cause dental disease [[#bib32|[32]]]  and [[#bib33|[33]]] .      

==MicroRNAs as biomarkers for oral cancers==

MicroRNAs are important in tumourigenesis due to their proximity to chromosomal breakpoints and their dysregulated expression levels in many malignancies [[#bib34|[34]]] , [[#bib35|[35]]]  and [[#bib36|[36]]] . Overexpression of certain miRNAs might result in the downregulation of tumour suppressor genes, while underexpression of other miRNAs might cause oncogene upregulation [[#bib37|[37]]]  and [[#bib38|[38]]] . Consequently, several studies evaluated the potential of miRNAs as diagnostic and prognostic biomarkers for cancers [[#bib20|[20]]] , [[#bib34|[34]]] , [[#bib39|[39]]]  and [[#bib40|[40]]] .      

Approximately 90% of head and neck cancers are oral squamous cell carcinomas (OSCC). OSCC is an aggressive and lethal malignancy that is a major worldwide problem due to its extremely high prevalence and very poor prognosis [[#bib41|[41]]]  and [[#bib42|[42]]] . The average 5-year survival rate for patients with diagnosed OSCC is approximately 50% [[#bib43|[43]]]  and [[#bib44|[44]]] . Therefore, an early detection method is needed for OSCC to improve treatment and long-term patient survival [[#bib20|[20]]] . A number of studies have evaluated miRNAs as diagnostic biomarkers for OSCC. Differentially expressed miRNAs discovered by these studies are listed in [[#t0005|Table 1]] .

<span id='t0005'></span>

{| class="wikitable" style="min-width: 60%;margin-left: auto; margin-right: auto;"
|+

Table 1.

Differential expression of miRNAs in oral cancers (Human model).

|-

! miRNA
! Sample source
! Disease
! Expression
! Reference
|-

! miR-211
| Tissue
| OSCC
| Increased
| Chang et al. [[#bib45|[45]]]
|-

! miR-125a, miR-200a
| Saliva
| OSCC
| Increased
| Park et al. [[#bib20|[20]]]
|-

! miR-21, miR-155, let-7i, miR142-3p, miR-423, miR-106b, miR-20a, miR-16
| rowspan="2" | Tissue
| rowspan="2" | HNSCC
| Increased
| rowspan="2" | Hui et al. [[#bib46|[46]]]
|-

! miR-125b, miR-375, miR-10a
| Decreased
|-

! miR-24
| Plasma
| OSCC
| Increased
| Lin et al. [[#bib47|[47]]]
|-

! miR-31
| Plasma
| OSCC
| Increased
| Liu et al. [[#bib22|[22]]]
|-

! miR-99a, miR-100
| Tissue
| HNSCC
| Decreased
| Chen et al. [[#bib48|[48]]]
|-

! miR-9
| rowspan="2" | Saliva
| rowspan="2" | HNSCC
| Increased
| rowspan="2" | Salazar et al. [[#bib49|[49]]]
|-

! miR-134, miR-191
| Decreased
|-

! miR-21
| Whole blood
| OSCC
| Increased
| Ren et al. [[#bib50|[50]]]
|-

! miR-155
| Tissue
| OSCC
| Increased
| Shi et al. [[#bib51|[51]]]
|-

! miR-27b
| Saliva
| OSCC
| Increased
| Momen-Heravi et al. [[#bib38|[38]]]
|}

* Head and neck squamous cell carcinoma (HNSCC), oral squamous cell carcinoma (OSCC).

==MicroRNAs as biomarkers for periodontitis==

Periodontal disease is characterised by inflammation of tooth-supporting structures. Periodontitis, which is a typical manifestation of periodontal disease, involves progressive loss of the alveolar bone around the teeth. If left untreated, periodontitis can lead to tooth loosening and subsequent tooth loss [[#bib52|[52]]] , [[#bib53|[53]]] , [[#bib54|[54]]]  and [[#bib55|[55]]] . Associations between periodontal disease and other general health problems, such as diabetes, cardiovascular disease, rheumatoid arthritis and adverse pregnancy outcome, have been reported in recent years, indicating the importance of early detection and treatment of periodontal disease [[#bib56|[56]]] . The development of diagnostic tools to detect the presence and activity of periodontal disease is therefore of high importance.      

Periodontal disease severity has traditionally been assessed using clinical parameters like pocket probing depth, clinical attachment loss, bleeding on probing, and radiographic determination of alveolar bone loss. Most of these techniques were established more than five decades ago, and lack the capacity to identify highly susceptible patients at risk for disease progression [[#bib28|[28]]]  and [[#bib57|[57]]] . Periodontitis is a highly complex disease, which hampers the development of rapid, accurate, diagnostic and prognostic tests. Nevertheless, the development of innovative diagnostic tests for periodontal disease remains a high priority.      

A small number of miRNA studies related to periodontal disease have been performed ([[#t0010|Table 2]] ). Xie et al. [[#bib58|[58]]]  used microarray analysis to examine miRNA expression, and transcript levels of selected inflammatory-related miRNAs were confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The study used gingival tissues from ten healthy subjects and ten patients with periodontitis. Levels of some miRNAs were more than five-fold higher in tissues from periodontitis patients than in control tissues. In addition, the possible regulation of Toll-lik receptors (TLRs) in periodontal inflammation by miRNA pathways was also proposed.

<span id='t0010'></span>

{| class="wikitable" style="min-width: 60%;margin-left: auto; margin-right: auto;"
|+

Table 2.

MicroRNA research related to periodontal disease.

|-

! Target diseases
! Sample source
! Methods
! Results
! Reference
|-

! Periodontitis
| Healthy and diseased gingival tissues
| miRNA PCR array
| 6 miRNAs up-regulated
| Lee et al. [[#bib64|[64]]]
|-

! Periodontitis
| Healthy and diseased gingival tissues
| miRNA Microarray
| 91 miRNAs up-regulated, 34 miRNAs down-regulated
| Xie et al. [[#bib58|[58]]]
|-

! Periodontitis and obesity
| Gingival biopsy samples
| miRNA PCR array
| 11 miRNAs up-regulated
| Perri et al. [[#bib59|[59]]]
|-

! Periodontitis
| Healthy and diseased gingival tissues
| miRNA Microarray
| 4 miRNAs up-regulated, 7 miRNAs down-regulated
| Stoecklin-Wasmer et al. [[#bib61|[61]]]
|}

A pilot investigation was conducted to determine whether miRNA expression was altered by obesity or periodontal disease, and whether there were any potential interactions between obesity and periodontitis that could involve miRNA modulation [[#bib59|[59]]] . In this study, gingival biopsy samples were obtained from 20 patients, ten of who were non-obese (BMI<30 kg/m<sup>2</sup> ) and ten of who were obese (BMI>30 kg/m<sup>2</sup> ). Each group of ten patients contained five patients with chronic periodontitis and five periodontally healthy patients. This was the first trial to assess the mechanisms underlying the pathogenesis of periodontitis and a common chronic condition (obesity), as well as the interaction between the two diseases [[#bib60|[60]]] .      

Stoecklin-Wasmer et al. [[#bib61|[61]]]  examined the occurrence of miRNAs in healthy and diseased gingival tissues and validated the in silico-predicted targets through mRNA profiling using whole-genome microarray analysis of the same specimens. Four miRNAs were significantly overexpressed, and seven significantly underexpressed, in gingival tissues compared to controls. Gene Set Enrichment Analysis (GSEA) identified 60 enriched miRNA gene sets with target genes involved in immune/inflammatory responses and tissue homeostasis. This was the first study to examine concurrent mRNA and miRNA expression in the same gingival tissues.      

Only a handful of studies investigating miRNAs in gingival tissues have been reported to date. Studies of miRNAs and their relationships to periodontal disease will be improved in the near future by the use of diverse samples, including saliva, and this will allow the inter-relationships of periodontal disease with other systemic diseases to be ascertained. In addition, some studies using animal periodontitis models and dental stem cells have been conducted [[#bib62|[62]]]  and [[#bib63|[63]]] , and these will help determine the mechanisms underlying the modulation of specific candidate miRNAs.      

==Limitations of miRNAs as biomarkers==

Although the exploitation of miRNAs as biomarkers for dental diseases is promising, some constraints should be considered.

First, differentially expressed candidate miRNAs identified through pilot studies need to be validated. Most previous studies using tissues were performed with samples from small numbers of individuals without matching for potential confounding factors known to influence periodontitis susceptibility such as age and gender. Further validation studies using large well-characterised cohorts are required [[#bib60|[60]]] .      

Second, although recent advances in molecular biology and high-throughput screening techniques have enabled researchers to characterise miRNA patterns in body fluids such as serum, plasma, and saliva on a large scale, this is limited by the lack of suitable endogenous controls for normalisation of salivary miRNAs. Recent research attempted to identify endogenous control miRNAs displaying minimal expression variability between samples [[#bib38|[38]]] , but endogenous salivary miRNA controls are required for future exploitation of miRNA datasets. In addition, saliva samples collected from the same individual can display considerable heterogeneity according to the collection method used, and standardised methods for sample collection should therefore be considered.      

Third, although biomarker development is paramount, the development of suitable treatment and prevention methods for patients testing positive for these biomarkers is also required. Effective, focused treatments are needed for highly susceptible patients in order to capitalise on early diagnosis, and without such treatments a clear cost-benefit advantage cannot be realised [[#bib28|[28]]] .      

==Future directions==

The availability of novel genetic testing technologies such as RNA sequencing will allow current technical limitations to be circumvented and will increase test accuracy when extremely small samples are used. Fundamental research into the mechanisms underlying control and activity of miRNAs will facilitate the identification of links between various diseases. In addition, more efficient computational prediction models and improved bioinformatic pipelines will allow optimal use of miRNA datasets [[#bib65|[65]]] , [[#bib66|[66]]]  and [[#bib67|[67]]] .      

The combination of new diagnostic tests with saliva sampling will provide easy, rapid, testing, and will enable large scale and follow-up studies to be conducted at lower costs than when using traditional blood or tissue samples. With the introduction of new and improved techniques, more individuals susceptible to periodontal disease and with poor prognosis for other conditions will be detected early, allowing patient-focused clinical treatments. This first step will lay the groundwork for the future development of personalised dentistry using genetic information.

==Acknowledgements==

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning  ([[#gs2|NRF-2013R1A1A2011974]] ).

==References==

<ol style='list-style-type: none;margin-left: 0px;'><li><span id='bib1'></span>
[[#bib1|[1]]] R.C. Lee, R.L. Feinbaum, V. Ambros; The ''C'' . ''elegans''  heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14                                        ; Cell, 75 (1993), pp. 843–854</li>
<li><span id='bib2'></span>
[[#bib2|[2]]] A.E. Pasquinelli, B.J. Reinhart, F. Slack, M.Q. Martindale, M.I. Kuroda, B. Maller,  ''et al.''; Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA; Nature, 408 (2000), pp. 86–89</li>
<li><span id='bib3'></span>
[[#bib3|[3]]] M. Weiland, X.H. Gao, L. Zhou, Q.S. Mi; Small RNAs have a large impact: circulating microRNAs as biomarkers for human diseases; RNA Biol., 9 (2012), pp. 850–859</li>
<li><span id='bib4'></span>
[[#bib4|[4]]] D.P. Bartel; MicroRNAs: target recognition and regulatory functions; Cell, 136 (2009), pp. 215–233</li>
<li><span id='bib5'></span>
[[#bib5|[5]]] M. Selbach, B. Schwanhausser, N. Thierfelder, Z. Fang, R. Khanin, N. Rajewsky; Widespread changes in protein synthesis induced by microRNAs; Nature, 455 (2008), pp. 58–63</li>
<li><span id='bib6'></span>
[[#bib6|[6]]] D.W. Thomson, C.P. Bracken, G.J. Goodall; Experimental strategies for microRNA target identification; Nucleic Acids Res., 39 (2011), pp. 6845–6853</li>
<li><span id='bib7'></span>
[[#bib7|[7]]] D. Baek, J. Villen, C. Shin, F.D. Camargo, S.P. Gygi, D.P. Bartel; The impact of microRNAs on protein output; Nature, 455 (2008), pp. 64–71</li>
<li><span id='bib8'></span>
[[#bib8|[8]]] A. Kozomara, S. Griffiths-Jones; miRBase: annotating high confidence microRNAs using deep sequencing data; Nucleic Acids Res., 42 (2014), pp. D68–D73</li>
<li><span id='bib9'></span>
[[#bib9|[9]]] R. Dai, S.A. Ahmed; MicroRNA, a new paradigm for understanding immunoregulation, inflammation, and autoimmune diseases; Trans. Res.: J. Lab. Clin. Med., 157 (2011), pp. 163–179</li>
<li><span id='bib10'></span>
[[#bib10|[10]]] Y. Lee, C. Ahn, J. Han, H. Choi, J. Kim, J. Yim,  ''et al.''; The nuclear RNase III Drosha initiates microRNA processing; Nature, 425 (2003), pp. 415–419</li>
<li><span id='bib11'></span>
[[#bib11|[11]]] H.K. Saini, A.J. Enright, S. Griffiths-Jones; Annotation of mammalian primary microRNAs; BMC Genom., 9 (2008), p. 564</li>
<li><span id='bib12'></span>
[[#bib12|[12]]] E. Bernstein, A.A. Caudy, S.M. Hammond, G.J. Hannon; Role for a bidentate ribonuclease in the initiation step of RNA interference; Nature, 409 (2001), pp. 363–366</li>
<li><span id='bib13'></span>
[[#bib13|[13]]] D.P. Bartel; MicroRNAs: genomics, biogenesis, mechanism, and function; Cell, 116 (2004), pp. 281–297</li>
<li><span id='bib14'></span>
[[#bib14|[14]]] D.S. Schwarz, G. Hutvagner, B. Haley, P.D. Zamore; Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways; Mol. Cell, 10 (2002), pp. 537–548</li>
<li><span id='bib15'></span>
[[#bib15|[15]]] X. Chen, Y. Ba, L. Ma, X. Cai, Y. Yin, K. Wang,  ''et al.''; Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases; Cell Res., 18 (2008), pp. 997–1006</li>
<li><span id='bib16'></span>
[[#bib16|[16]]] P.S. Mitchell, R.K. Parkin, E.M. Kroh, B.R. Fritz, S.K. Wyman, E.L. Pogosova-Agadjanyan,  ''et al.''; Circulating microRNAs as stable blood-based markers for cancer detection; Proc. Natl. Acad. Sci. USA, 105 (2008), pp. 10513–10518</li>
<li><span id='bib17'></span>
[[#bib17|[17]]] J.P. Duong Van Huyen, M. Tible, A. Gay, R. Guillemain, O. Aubert, S. Varnous,  ''et al.''; MicroRNAs as non-invasive biomarkers of heart transplant rejection; Eur. Heart J., 35 (45) (2014), pp. 3194–3202</li>
<li><span id='bib18'></span>
[[#bib18|[18]]] C.H. Lawrie, S. Gal, H.M. Dunlop, B. Pushkaran, A.P. Liggins, K. Pulford,  ''et al.''; Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma; Br. J. Haematol., 141 (2008), pp. 672–675</li>
<li><span id='bib19'></span>
[[#bib19|[19]]] Y. Li, X. Zhou St, M.A. John, D.T. Wong; RNA profiling of cell-free saliva using microarray technology; J. Dent. Res., 83 (2004), pp. 199–203</li>
<li><span id='bib20'></span>
[[#bib20|[20]]] N.J. Park, H. Zhou, D. Elashoff, B.S. Henson, D.A. Kastratovic, E. Abemayor,  ''et al.''; Salivary microRNA: discovery, characterization, and clinical utility for oral cancer detection; Clin. Cancer Res., 15 (2009), pp. 5473–5477</li>
<li><span id='bib21'></span>
[[#bib21|[21]]] R.S. Patel, A. Jakymiw, B. Yao, B.A. Pauley, W.C. Carcamo, J. Katz,  ''et al.''; High resolution of microRNA signatures in human whole saliva; Arch. Oral Biol., 56 (2011), pp. 1506–1513</li>
<li><span id='bib22'></span>
[[#bib22|[22]]] C.J. Liu, S.C. Lin, C.C. Yang, H.W. Cheng, K.W. Chang; Exploiting salivary miR-31 as a clinical biomarker of oral squamous cell carcinoma; Head Neck, 34 (2012), pp. 219–224</li>
<li><span id='bib23'></span>
[[#bib23|[23]]] J.M. Yoshizawa, D.T. Wong; Salivary microRNAs and oral cancer detection; Methods Mol. Biol., 936 (2013), pp. 313–324</li>
<li><span id='bib24'></span>
[[#bib24|[24]]] X. Lin, H.C. Lo, D.T. Wong, X. Xiao; Noncoding RNAs in human saliva as potential disease biomarkers; Front. Genet., 6 (2015), p. 175 [http://dx.doi.org/10.3389/fgene.2015.00175 http://dx.doi.org/10.3389/fgene.2015.00175]</li>
<li><span id='bib25'></span>
[[#bib25|[25]]] J.A. Webber, D.H. Baxter, S. Zhang, D.Y. Huang, K.H. Huang, M.J. Lee,  ''et al.''; The microRNA spectrum in 12 body fluids; Clin. Chem., 56 (2010), pp. 1733–1741</li>
<li><span id='bib26'></span>
[[#bib26|[26]]] R.J. Genco; Salivary diagnostic tests; J. Am. Dent. Assoc., 143 (2012), pp. 3S–5S</li>
<li><span id='bib27'></span>
[[#bib27|[27]]] W.V. Giannobile, D.T. Wong; Salivary diagnostics: oral health and beyond!; J. Dent. Res., 90 (2011), pp. 1153–1154</li>
<li><span id='bib28'></span>
[[#bib28|[28]]] W.V. Giannobile, T. Beikler, J.S. Kinney, C.A. Ramseier, T. Morelli, D.T. Wong; Saliva as a diagnostic tool for periodontal disease: current state and future directions; Periodontology, 50 (2000), pp. 52–64 2009</li>
<li><span id='bib29'></span>
[[#bib29|[29]]] S. Bonassi, M. Neri, R. Puntoni; Validation of biomarkers as early predictors of disease; Mutat. Res., 480–481 (2001), pp. 349–358</li>
<li><span id='bib30'></span>
[[#bib30|[30]]] H. Fabryova, P. Celec; On the origin and diagnostic use of salivary RNA; Oral Dis., 20 (2014), pp. 146–152</li>
<li><span id='bib31'></span>
[[#bib31|[31]]] N. Christodoulides, P.N. Floriano, C.S. Miller, J.L. Ebersole, S. Mohanty, P. Dharshan,  ''et al.''; Lab-on-a-chip methods for point-of-care measurements of salivary biomarkers of periodontitis; Ann. N. Y. Acad. Sci., 1098 (2007), pp. 411–428</li>
<li><span id='bib32'></span>
[[#bib32|[32]]] D. Malamud; Salivary diagnostics: the future is now; J. Am. Dent. Assoc., 137 (284) (2006), p. 286</li>
<li><span id='bib33'></span>
[[#bib33|[33]]] W.V. Giannobile, J.T. McDevitt, R.S. Niedbala, D. Malamud; Translational and clinical applications of salivary diagnostics; Adv. Dent. Res., 23 (2011), pp. 375–380</li>
<li><span id='bib34'></span>
[[#bib34|[34]]] T.A. Farazi, J.I. Spitzer, P. Morozov, T. Tuschl; miRNAs in human cancer. The; J. Pathol., 223 (2011), pp. 102–115</li>
<li><span id='bib35'></span>
[[#bib35|[35]]] J. Lu, G. Getz, E.A. Miska, E. Alvarez-Saavedra, J. Lamb, D. Peck,  ''et al.''; MicroRNA expression profiles classify human cancers; Nature, 435 (2005), pp. 834–838</li>
<li><span id='bib36'></span>
[[#bib36|[36]]] G.A. Calin, C. Sevignani, C.D. Dumitru, T. Hyslop, E. Noch, S. Yendamuri,  ''et al.''; Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers; Proc. Natl. Acad. Sci. United States Am., 101 (2004), pp. 2999–3004</li>
<li><span id='bib37'></span>
[[#bib37|[37]]] C.Z. Chen; MicroRNAs as oncogenes and tumor suppressorshe; N. Engl. J. Med., 353 (2005), pp. 1768–1771</li>
<li><span id='bib38'></span>
[[#bib38|[38]]] F. Momen-Heravi, A.J. Trachtenberg, W.P. Kuo, Y.S. Cheng; Genomewide study of salivary microRNAs for detection of oral cancer; J. Dent. Res., 93 (2014), pp. 86S–93S</li>
<li><span id='bib39'></span>
[[#bib39|[39]]] Y. Wang, Q. Wang, N. Zhang, H. Ma, Y. Gu, H. Tang,  ''et al.''; Identification of microRNAs as novel biomarkers for detecting esophageal squamous cell carcinoma in Asians: a meta-analysis; Tumour Biol., 35 (2014), pp. 11595–11604</li>
<li><span id='bib40'></span>
[[#bib40|[40]]] N. Yanaihara, N. Caplen, E. Bowman, M. Seike, K. Kumamoto, M. Yi,  ''et al.''; Unique microRNA molecular profiles in lung cancer diagnosis and prognosis; Cancer Cell, 9 (2006), pp. 189–198</li>
<li><span id='bib41'></span>
[[#bib41|[41]]] T.J. Chiu, Y.J. Chen, K.M. Rau, C.H. Chen, C.Y. Chien, S.H. Li,  ''et al.''; Midkine neurite growth-promoting factor 2 expression as a potential prognostic marker of adjuvant therapy in head and neck squamous cell carcinoma; Biomarkers, 18 (2013), pp. 687–698</li>
<li><span id='bib42'></span>
[[#bib42|[42]]] A.K. Markopoulos; Current aspects on oral squamous cell carcinoma; Open Dent. J., 6 (2012), pp. 126–130</li>
<li><span id='bib43'></span>
[[#bib43|[43]]] B.M. Brinkman, D.T. Wong; Disease mechanism and biomarkers of oral squamous cell carcinoma; Curr. Opin. Oncol., 18 (2006), pp. 228–233</li>
<li><span id='bib44'></span>
[[#bib44|[44]]] G.F. Funk, L.H. Karnell, R.A. Robinson, W.K. Zhen, D.K. Trask, H.T. Hoffman; Presentation, treatment, and outcome of oral cavity cancer: a National Cancer Data Base report; Head Neck, 24 (2002), pp. 165–180</li>
<li><span id='bib45'></span>
[[#bib45|[45]]] K.W. Chang, C.J. Liu, T.H. Chu, H.W. Cheng, P.S. Hung, W.Y. Hu,  ''et al.''; Association between high miR-211 microRNA expression and the poor prognosis of oral carcinoma; J. Dent. Res., 87 (2008), pp. 1063–1068</li>
<li><span id='bib46'></span>
[[#bib46|[46]]] A.B. Hui, M. Lenarduzzi, T. Krushel, L. Waldron, M. Pintilie, W. Shi,  ''et al.''; Comprehensive MicroRNA profiling for head and neck squamous cell carcinomas; Clin. Cancer Res., 16 (2010), pp. 1129–1139</li>
<li><span id='bib47'></span>
[[#bib47|[47]]] S.C. Lin, C.J. Liu, J.A. Lin, W.F. Chiang, P.S. Hung, K.W. Chang; miR-24 up-regulation in oral carcinoma: positive association from clinical and in vitro analysis; Oral Oncol., 46 (2010), pp. 204–208</li>
<li><span id='bib48'></span>
[[#bib48|[48]]] Z. Chen, Y. Jin, D. Yu, A. Wang, I. Mahjabeen, C. Wang,  ''et al.''; Down-regulation of the microRNA-99 family members in head and neck squamous cell carcinoma; Oral Oncol., 48 (2012), pp. 686–691</li>
<li><span id='bib49'></span>
[[#bib49|[49]]] C. Salazar, R. Nagadia, P. Pandit, J. Cooper-White, N. Banerjee, N. Dimitrova,  ''et al.''; A novel saliva-based microRNA biomarker panel to detect head and neck cancers; Cell. Oncol., 37 (2014), pp. 331–338</li>
<li><span id='bib50'></span>
[[#bib50|[50]]] W. Ren, C. Qiang, L. Gao, S.M. Li, L.M. Zhang, X.L. Wang,  ''et al.''; Circulating microRNA-21 (MIR-21) and phosphatase and tensin homolog (PTEN) are promising novel biomarkers for detection of oral squamous cell carcinoma; Biomarkers, 19 (7) (2014), pp. 590–596</li>
<li><span id='bib51'></span>
[[#bib51|[51]]] L.J. Shi, C.Y. Zhang, Z.T. Zhou, J.Y. Ma, Y. Liu, Z.X. Bao,  ''et al.''; MicroRNA-155 in oral squamous cell carcinoma: overexpression, localization, and prognostic potential; Head neck, 37 (2014), pp. 970–976</li>
<li><span id='bib52'></span>
[[#bib52|[52]]] G.C. Armitage; Clinical evaluation of periodontal diseases; Periodontology, 7 (2000), pp. 39–53 1995</li>
<li><span id='bib53'></span>
[[#bib53|[53]]] G.C. Armitage, P.B. Robertson; The biology, prevention, diagnosis and treatment of periodontal diseases: scientific advances in the United States; J. Am. Dent. Assoc., 140 (Suppl. 1) (2009), pp. 36S–43S</li>
<li><span id='bib54'></span>
[[#bib54|[54]]] S.H. Kim, B.M. Seo, P.H. Choung, Y.M. Lee; Adult stem cell therapy for periodontal disease; Int. J. Stem Cells, 3 (2010), pp. 16–21</li>
<li><span id='bib55'></span>
[[#bib55|[55]]] S. Offenbacher; Periodontal diseases: pathogenesis; Ann. Periodontol./Am. Acad. Periodontol., 1 (1996), pp. 821–878</li>
<li><span id='bib56'></span>
[[#bib56|[56]]] D. Kinane, P. Bouchard,  Group EoEWoP; Periodontal diseases and health: consensus report of the sixth European workshop on periodontology; J. Clin. Periodontol., 35 (2008), pp. 333–337</li>
<li><span id='bib57'></span>
[[#bib57|[57]]] J.M. Goodson; Diagnosis of periodontitis by physical measurement: interpretation from episodic disease hypothesis; J. Periodontol., 63 (1992), pp. 373–382</li>
<li><span id='bib58'></span>
[[#bib58|[58]]] Y.F. Xie, R. Shu, S.Y. Jiang, D.L. Liu, X.L. Zhang; Comparison of microRNA profiles of human periodontal diseased and healthy gingival tissues; Int. J. Oral Sci., 3 (2011), pp. 125–134</li>
<li><span id='bib59'></span>
[[#bib59|[59]]] R. Perri, S. Nares, S. Zhang, S.P. Barros, S. Offenbacher; MicroRNA modulation in obesity and periodontitis; J. Dent. Res., 91 (2012), pp. 33–38</li>
<li><span id='bib60'></span>
[[#bib60|[60]]] F. D׳Aiuto, J. Suvan; Obesity, inflammation, and oral infections: are microRNAs the missing link?; J. Dent. Res., 91 (2012), pp. 5–7</li>
<li><span id='bib61'></span>
[[#bib61|[61]]] C. Stoecklin-Wasmer, P. Guarnieri, R. Celenti, R.T. Demmer, M. Kebschull, P.N. Papapanou; MicroRNAs and their target genes in gingival tissues; J. Dent. Res., 91 (2012), pp. 934–940</li>
<li><span id='bib62'></span>
[[#bib62|[62]]] M.A. Nahid, M. Rivera, A. Lucas, E.K. Chan, L. Kesavalu; Polymicrobial infection with periodontal pathogens specifically enhances microRNA miR-146a in ApoE-/- mice during experimental periodontal disease; Infect. Immun., 79 (2011), pp. 1597–1605</li>
<li><span id='bib63'></span>
[[#bib63|[63]]] Y. Liu, W. Liu, C. Hu, Z. Xue, G. Wang, B. Ding,  ''et al.''; MiR-17 modulates osteogenic differentiation through a coherent feed-forward loop in mesenchymal stem cells isolated from periodontal ligaments of patients with periodontitis; Stem Cells, 29 (2011), pp. 1804–1816</li>
<li><span id='bib64'></span>
[[#bib64|[64]]] Y.H. Lee, H.S. Na, S.Y. Jeong, S.H. Jeong, H.R. Park, J. Chung; Comparison of inflammatory microRNA expression in healthy and periodontitis tissues; Biocell, 35 (2011), pp. 43–49</li>
<li><span id='bib65'></span>
[[#bib65|[65]]] S.Y. Lee, K.A. Sohn, J.H. Kim; MicroRNA-centric measurement improves functional enrichment analysis of co-expressed and differentially expressed microRNA clusters; BMC Genom., 13 (Suppl 7) (2012), p. S17</li>
<li><span id='bib66'></span>
[[#bib66|[66]]] E. Sonkoly, A. Pivarcsi; microRNAs in inflammation; Int. Rev. Immunol., 28 (2009), pp. 535–561</li>
<li><span id='bib67'></span>
[[#bib67|[67]]] A. Tiwari, S. Shivananda, K.S. Gopinath, A. Kumar; microRNA-125a reduces proliferation and invasion of oral squamous cell carcinoma cells by targeting estrogen-related receptor alpha: implications for cancer therapeutics; J. Biol. Chem., 289 (2014), pp. 32276–32290</li>
</ol>