Oral Diseases (2015) 21, 739–747 doi:10.1111/odi.12340 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd All rights reserved www.wiley.com

ORIGINAL ARTICLE

Salivary microRNAs in oral cancer F Zahran1,2, D Ghalwash3, O Shaker4, K Al-Johani1, C Scully5 1

Division of Oral Medicine, Oral Diagnostic Sciences Department, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia; 2Department of Oral Medicine and Periodontology, Faculty of Oral and Dental Medicine, Cairo University, Cairo; 3 Department of Oral Medicine and Periodontology, Faculty of Dentistry, October University for Modern Sciences and Arts, 6th October City; 4Department of Medical Biochemistry, Faculty of Medicine, Cairo University, Cairo, Egypt; 5Emeritus Professor, UCL, London, UK

OBJECTIVE: This study investigated the use of three salivary microRNAs (miRNA-21, miRNA-184, and miRNA-145) as possible markers for malignant transformation in oral mucosal lesions. MATERIALS AND METHODS: Salivary whole unstimulated samples were collected from a study group of 100 subjects, consisting of 20 clinically healthy controls, 40 patients with oral potentially malignant disorders (PMDs) [20 with dysplastic lesions and 20 without dysplasia], 20 with biopsy-confirmed oral squamous cell carcinoma (OSCC), and 20 with recurrent aphthous stomatitis (RAS) as disease controls. Total RNA was isolated and purified from saliva samples using the microRNA Isolation Kit (Qiagen, UL). miRNA expression analysis was performed using qRT-PCR (Applied Biosystems). RESULTS: There was a highly significant increase in salivary miRNA-21 and miRNA-184 in OSCC and PMD (with and without dysplasia) when compared to healthy and disease controls (P < 0.001). Conversely, miRNA145 levels showed a highly significant decrease in OSCC and PMD overall (P < 0.001). RAS cases showed no significant difference from normal controls in any measured miRNA (P > 0.05). The only microRNA to discriminate between OSCC and PMD with dysplasia was miRNA-184. When receiver operating characteristic curves were designed for the three miRNAs, cutoff points delineating the occurrence of malignant change were a fourfold increase in miRNA-21 with specificity 65% and sensitivity 65%, a 0.6 decrease in miRNA-145, with specificity 70% and sensitivity 60%, and a threefold increase of miRNA-184, with specificity 75% and sensitivity 80%. Calculating the area under the curve revealed that miRNA-184 was the only one among the studied miRNAs that provided good diagnostic value. Correspondence: Fat’heya Zahran, Division of Oral Medicine, Oral Diagnostic Sciences Department, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia. Tel: +966556412275, Fax: +966 012 6403316, E-mail: [email protected] Received 2 October 2014; revised 26 February 2015; accepted 10 March 2015

CONCLUSION: Salivary determination of the miRNAs tested might furnish a noninvasive, rapid adjunctive aid for revealing malignant transformation in oral mucosal lesions, particularly miRNA-184. Oral Diseases (2015) 21, 739–747 Keywords: oral cancer; microRNA; miRNA-21; miRNA-145; miRNA-184; miRNA salivary biomarkers; potentially malignant disorders; oral malignant transformation

Introduction Oral squamous cell carcinoma (OSCC) is the 6th most frequent cancer worldwide (Jemal et al, 2011). The poor prognosis has led to efforts to try to clarify the mechanisms underlying the high invasiveness and to investigate new diagnostic and therapeutic strategies (Yanamoto et al, 2002; Kawakita et al, 2013). Diagnostic and prognostic biomarkers have been sought, and as deregulation of microRNAs (miRNAs) has been shown to correlate with various tumor characteristics and prognosis in some cancers, including those affecting the oral cavity (Calin and Croce, 2006; Wu et al, 2011; Chen et al, 2013), this was considered to be a fruitful area to explore. Like other cancers, oral carcinogenesis involves gradual accumulation of both genetic and epigenetic changes, leading to gain of function in certain oncogenes and loss of function in some tumor suppressor genes (Leemans et al, 2011). miRNAs are small noncoding RNAs which function in mRNA silencing and posttranscriptional regulation of gene expression. miRNAs are key regulators (Grosshans and Slack, 2002) transcribed by RNA polymerase II or RNA polymerase III as a part of an intron of mRNA or as an independent gene unit. Both whole and supernatant saliva of healthy controls contain many miRNAs which enter the oral cavity through various sources, including the salivary glands, gingival crevicular fluid, and from desquamated oral epithelial cells (Park et al, 2006). Most salivary miRNAs are partially degraded (Park et al, 2007) and maintain stability in saliva

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through their association with unidentified macromolecules (Park et al, 2006). Deregulation of miRNAs is known to be associated with many diseases, and as salivary miRNAs are stable, they have been suggested to have potential use in oral cancer detection (Park et al, 2009). One group very active in the field detected
50 miRNAs in both whole and supernatant saliva, although whole saliva contained a more heterogeneous population of miRNAs; two salivary miRNAs, miRNA-200a and miRNA125a, were present at lower levels in saliva of patient with OSCC than in healthy controls (Park et al, 2009). miRNAs may be discriminatory in patients with OSCC (Momen-Heravi et al, 2014) and have been studied in oral potentially malignant diseases (PMDs) (Roy et al, 2014) as well as in cell models and OSCC (Severino et al, 2013; Yoshizawa and Wong, 2013). miRNAs overexpressed and deregulated in squamous cell carcinomas of the head and neck include miRNA-1, miRNA-7, miRNA-10b, and miRNA-196a (Andreghetto et al, 2013). Investigation of genomewide expression patterns of salivary miRNAs using NanoString nCounter miRNA expression assay and real-time quantitative PCR (qPCR), followed by construction of receiver operating characteristic (ROC) curves to determine the sensitivity and specificity of the assay on more than 700 miRNAs, identified 13 miRNAs as being significantly deregulated in patients with OSCC. 11 miRNAs were underexpressed (miRNA-136, miRNA147, miRNA-1250, miRNA-148a, miRNA-632, miRNA646, miRNA-668, miRNA-877, miRNA-503, miRNA-220a, and miRNA-323-5p), and 2 miRNAs were overexpressed (miRNA-24 and miRNA-27b). Receiver operating characteristic curve analyses showed that miRNA-27b could be a valuable biomarker for distinguishing patients with OSCC from the other groups (Momen-Heravi et al, 2014). miRNA21 is upregulated in most cancers, including head and neck carcinomas (Fu et al, 2011), and it has been postulated that miRNA-21 expression might indicate a worse prognosis (Asangani et al, 2008; Hwang et al, 2010). miRNA-21 may have a role in invasion and metastasis via several target molecules (Reis et al, 2010; Han et al, 2012a; Zhang et al, 2012), and it may be implicated in survival (Hedb€ack et al, 2014). However, little is known about the mechanism by which miRNA-21 affects OSCC (Kawakita et al, 2013). miRNA-145, a major tumor suppressor miRNA, plays a pivotal role in regulating apoptosis (Ostenfeld et al, 2010) as well as being downregulated in many cancers, including those of the prostate, bladder, and colon (Akao et al, 2007; Ozen et al, 2008; Ichimi et al, 2009). Downregulation of miRNA-145 has been demonstrated in an animal model of OSCC (Yu et al, 2009). miRNA-184 may target several hundred genes (Weitzel et al, 2009; Foley et al, 2010; Liu et al, 2011) and inhibits neuroblastoma cell survival through targeting AKT2, the serine/threonine kinase. However, few studies have investigated miRNA-184 expression as a risk factor for cancer (Liu et al, 2011) although Yu et al (2008) observed that miRNA-184 had a tumor-suppressive effect on squamous cell carcinoma (SCC) cell lines. Wong et al (2008) showed that miRNA184 was important in the anti-apoptotic and proliferative processes in OSCC.

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Consequently, we elected to investigate the possible usefulness of these 3 miRNAs in whole saliva as biomarkers for oral mucosal dysplasia and malignant transformation.

Materials and methods The study received ethical approval from King Abdulaziz University, Jeddah, Saudi Arabia (no. 078-13, date: 15/12/2013). All included subjects signed a consent form denoting their agreement to participate.

Study groups All included individuals were ethnically Arabs; 100 subjects, selected from the outpatient clinic of Oral Medicine and Periodontology Department, Faculty of Oral and Dental Medicine, Cairo University and National Cancer Institute in Cairo, were categorized as: Group I. 20 healthy control subjects, with no significant oral or systemic disease. The exclusion of oral conditions included periodontal disease. Groups II and III. 40 patients with histologically confirmed PMD that had not transformed to OSCC over at least a 3-year period (Mishra et al, 2005; Joshi and Durve, 2007). According to the WHO classification system (Gale et al, 2005), this group included 20 patients with dysplastic PMD lesions (group II) and 20 others with PMD without dysplasia (group III). Group IV. 20 patients suffering from OSCC. All included subjects had newly diagnosed untreated primary tumors in the oral cavity. Group V. 20 patients suffering from recurrent aphthous stomatitis (RAS) were included as disease controls.

Salivary sample collection Collection of whole unstimulated saliva (WUS) using standard techniques was carried out as described by Navazesh (1993). Briefly, subjects refrained from eating, drinking, using chewing gum, etc., for at least one and a half hour prior to the evaluation. Samples were obtained by requesting subjects to swallow first, tilt their head forward, and expectorate all saliva in a tube for 5 min without swallowing. Samples were collected starting from December 2013 until June 2014.

RNA extraction We collected 5 ml saliva in sterilized tube and centrifuged at 2500 g for 10 min at 4°C. The supernatant was then collected and centrifuged at 10 000 g for 1 min to remove remaining cells. The resultant supernatant was transferred into a new tube, and RNA extraction was processed immediately. Soluble miRNA in supernatant (200 ll) was isolated using the miRNeasy serum/plasma extraction kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Extracted RNA was quantitated using NanoDropâ (ND)-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA), and then, cDNA was generated immediately by miScript RTII kit (Qiagen) according to the manufacturer’s recommendations.

miRNA extraction details This was performed using 1 ml QIAzol lysis reagent and incubated for 5 min at room temperature (20°C). Then, 200 ll chloroform was added, vortexed for 15 s, and incubated for 2–3 min at room temperature. This was followed by centrifugation at 12 000 g at 4°C for 15 min. The upper watery phase was removed, and an equal volume of 100% ethanol was added. Each 700 ll of this mixture was placed in RNeasy mini spin column in 2-ml collection tube and centrifuged at 10 000 rpm at room temperature for 15 s. After the mixture had completely passed the column, 700 ll of buffer RWT was added to each column and again centrifuged at 7 500 g at room temperature for 15 s. 500 ll buffer RPE was added to the column and centrifuged at 10 000 rpm at room temperature for 15 s. After this, another 500 ll buffer RPE was added to the column and centrifuged at 1000 rpm at room temperature for 2 min. The column

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placed in a new collection tube was then centrifuged at full speed for 2 min. The column was transferred to new 1.5 ml collection tube, and 50 ll RNase-free water was pipetted directly onto the column and centrifuged for 1 min at 10 000 rpm to elute RNA.

Reverse transcription Reverse transcription (RT) was carried out on 5 ng of total RNA in a final volume of 20 ll RT reactions (incubated for 60 min at 37°C, 5 min at 95°C, and then maintained at 4°C) using the miRNeasy serum/plasma reverse transcription kit (Qiagen) according to the manufacturer’s instructions.

Microarray platform and quantitative polymerase chain reaction Expression of miRNAs—miRNA-21, miRNA-145, and miRNA-184— was evaluated by qRT-PCR analysis according to the manufacturer’s protocol. Relative miRNA levels were determined by DDCt using endogenous controls (SNORD68). Real-time PCR was performed using the miScript SYBR green PCR kit (Qiagen) according to the manufacturer’s instructions. For real time PCR, 5 ll of diluted RT products (cDNA template) was mixed with SYBER Green Master Mix (Qiagen) in a final volume of 25 ll and added to a custom 96-well plate miScript miRNA PCR arrays (Qiagen), enriched with miRNA forward and reverse-miRNA-specific primer. Each primer was used separately. The plate was sealed with optical thin-wall 8-cap strips. Real-time PCR reactions were performed using an Applied Biosystems 7500 Real Time PCR System (Foster City, CA, USA) with the following conditions: 95°C for 15 min, followed by 40 cycles at 94°C for 15 s, and 55°C for 30 s and 70°C for 34 s. The cycle threshold (CT) is defined as the number of cycles required for the fluorescent signal to cross the threshold in real-time PCR. Every run contained negative control to be sure that it is actual expression and not contamination. The high CT values of 40 were excluded. Expression of miRNAs was registered as DCt value. The DCt was calculated by subtracting the CT values of miRNA SNORD68 from the CT values of the target miRNAs. As there is an inverse correlation between DCt and miRNA expression level, lower DCt values denoted increased miRNA levels. The resulting normalized DCt values were used in calculating relative expression values using 2D(Ct), and these values are directly related to the miRNA expression levels. The 2DD (Ct) calculation was then used to determine the relative quantitative levels of individual miRNAs.

Statistical analysis Statistical analysis was performed by SPSS (version 17: SPSS Inc, Chicago, USA). Variables were described by the mean, standard deviation (s.d.), standard error (s.e.), the range (maximum–minimum), and 95% confidence interval for mean. One-way analysis of variance (ANOVA) Ftest was used for comparing the means of all groups. It was then followed by the Dunnett t-test for comparing each group to the control group. Also, Scheffe’s multiple comparison method was used to compare the means of each two groups. Significance level was considered at P < 0.05 (significant), while for P < 0.01 and P < 0.001, it was considered highly significant. Two-tailed tests were carried out throughout the analysis for all statistical tests. A receiver operating characteristic curve (ROC) was created for each studied microRNA to estimate a preliminary cutoff point for the salivary level of each. Also, area under the ROC curve (AUC) was calculated. Taking in consideration the rough guide for classifying the accuracy of a diagnostic test by the traditional academic point system (Mehdi et al, 2011): • • • • •

0.90–1.0 = excellent 0.80–0.90 = good 0.70–0.80 = fair 0.60–0.70 = poor 0.50–0.60 = fail

Results The 20 healthy control subjects were 11 females and nine males, ranging from 37 to 65 years old, with mean age of

51.1  9.3 years. The 40 patients with PMD were 18 females and 22 males, ranging from 35 to 65 years old with mean age of 54.2  9.7 years, The 20 patients with OSCC were 12 females and eight males, ranging from 38 to 73 years old, with mean age of 58  9.2 years. The 20 RAS cases were seven females and 13 males, ranging from 19 to 36 years old, with mean age of 28  7.3 years. Table 1 shows demographic data of patients with OSCC. Table 2 shows the descriptive statistics for all studied miRNAs in different groups. For miRNA-21, the healthy controls had a range from 1.0 to 1.3 and a mean value of 1.16  0.09, the PMD without dysplasia values ranged from 2.2 to 4.7 with a mean value of 3.31  0.66, PMDs with dysplasia range was from 3.7 to 5.1 with mean value 4.13  0.41, and OSCC samples showed a range from 3.2 to 7.5 with mean value 4.82  1.40 (the highest of all), whereas RAS cases (disease controls) had a range from 1.0 to 1.3 and mean value 1.17  0.09. For miRNA-145 levels, the range for the healthy control group was 1.0–1.3 with mean value 1.16  0.10, the PMD without dysplasia ranged from 0.4 to 0.8 with mean value 0.67  0.12, those with PMD and dysplasia had a range from 0.6 to 1.0 and a mean value 0.74  0.11, and the OSCC cases showed a range from 0.3 to 1.0 and a mean of 0.61  0.20 (the lowest of all), whereas RAS cases had a range from 1.0 to 1.3 and a mean of 1.13  0.10. Regarding miRNA-184, the range was from 1.0 to 1.3 with mean value 1.15  0.09 for healthy controls, the values ranged from 1.1 to 3.1 with mean value 2.17  0.6 for PMD without dysplasia, and the values ranged from 2.3 to 3.3 with mean value 2.84  0.34 for PMDs with dysplasia, while OSCC samples values ranged from 2.4 to 5.1 with mean value 3.67  0.84 and RAS cases had a range from 1.0 to 1.3 and a mean of 1.15  0.09 (Figure 1). The figure summarizes and compares the mean values for the 3 miRNAs in all groups studied and reveals clearly how the levels of the three tested miRNAs seem comparable in the healthy controls and disease controls (RAS), while great variation occurs in PMD (whether with

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Table 1 Demographic data of OSCC patients Age Mean  s.d. Range Gender Male Female Smoking Yes No Site Ant 2/3 of tongue Post. 1/3 of tongue Lower alveolar margin Retro-molar Buccal mucosa Floor of the mouth Pathological presentation High grade undifferentiated SCC with LN involvement Grade III (high grade) LN involvement Grade II, LN involvement (2 LN) Grade II, no LN involvement No record available

58  9.2 38–73 8 (40%) 12 (60%) 6 (30%) 14 (70%) 4 4 4 4 2 2

(20%) (20%) (20%) (20%) (10%) (10%)

2 (10%) 7 (35%) 1 (5%) 7 (35%) 3 (15%)

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Table 2 Descriptive statistics

micro RNA -145

micro RNA -184

micro RNA -21 7.0

Salivary level

6.0 5.0 4.0

2.84 2.17 1.16

1.15

1.16

1.0 0.0

3.67

3.31

3.0 2.0

4.82

4.13

Normal control

0.67

PMD without dysplasia

0.61

0.74

PMD with dysplasia

OSCC

or without dysplasia) and OSCC cases, with the variation from normal taking a gradual pattern from PMD without dysplasia to PMD with dysplasia to OSCC. From the figure, it appears clear that the mean values for the healthy control group and RAS patients are very close, whereas those for the other three groups have values away from the healthy control levels. It is also evident that the highest values for miRNA-21 and miRNA-184 are seen in OSCC, followed by the PMD with dysplasia group, followed by the PMD group without dysplasia. Conversely, miRNA-145 shows the lowest values in OSCC cases. Table 3 shows Dunnett t-test results, comparing each study group to the healthy controls. Statistical analysis showed a highly significant increase (P < 0.001) in salivary miRNA-21 and miRNA-184 in cases with OSCC and PMD (whether with dysplasia or without), when compared to healthy controls. On the other hand, disease controls (RAS cases) showed a nonsignificant difference (P > 0.05) from the healthy controls. For miRNA-145 levels conversely, there was a highly significant decrease (P < 0.001) in cases with OSCC and in PMD, whether with dysplasia or without, when compared to healthy controls, and again RAS cases showed nonsignificant difference (P > 0.05) from the healthy controls. The results Oral Diseases

1.17 1.15

RAS

1.13

Figure 1 Relative quantification of different types of microRNA in studied groups

also showed a highly significant increase (P < 0.001) in salivary miRNA-184 in cases with OSCC and PMD (whether with dysplasia or without), when compared to healthy controls. On the other hand, disease controls (RAS cases) showed a nonsignificant difference (P > 0.05) from the healthy controls. Table 4 shows the results when groups were compared statistically by Scheffe’s method. There was a nonsignificant difference (P > 0.05) between OSCC and PMDs with dysplasia regarding the levels of salivary miRNA-21 and miRNA-145, while miRNA-184 showed a highly significant difference (P < 0.001) between these two groups. On the other hand, there was a highly significant difference between OSCC and PMD without dysplasia regarding the levels of salivary miRNA-21 and miRNA-184 (P < 0.001), whereas there was a nonsignificant difference (P > 0.05) in miRNA-145 levels among these two groups. Also PMD with dysplasia was not statistically different (P > 0.05) from those without dysplasia regarding miRNA-145 levels. When the same two groups were compared regarding miRNA-21 and miRNA-184, the difference was statistically different (P < 0.05). The difference between RAS group and any of the former three groups was highly significant (P < 0.001) regarding the salivary levels of all studied miRNAs. What was remark-

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Table 3 Dunnett t-test comparing each study group to the healthy controls miRNA-21

Groups compared PMD without dysplasia PMD with dysplasia OSCC RAS

Control Control Control Control

miRNA-145

Mean differ-ence

Standard error

P-value

Mean differ-ence

2.155 2.975 3.660 0.010

0.228 0.228 0.228 0.228

0.00000b 0.00000b 0.00000b 1.00000c

0.485 0.415 0.545 0.025

Standard error 0.042 0.042 0.042 0.042

miRNA-184

P-value

Mean differ-ence

Standard error

P-value

0.00000b 0.00000b 0.00000b 0.93479c

1.020 1.695 2.520 0.005

0.155 0.155 0.155 0.155

0.00000b 0.00000b 0.00000b 1.00000c

P < 0.05 Significant. P < 0.001 Highly significant. c P > 0.05 Nonsignificant. a

b

Table 4 Scheffe’s method for comparing each two groups regarding salivary miRNA levels miRNA-21 Mean difference

Groups compared PMD without dysplasia PMD without dysplasia PMD without dysplasia PMD with dysplasia PMD with dysplasia OSCC

PMD with dysplasia OSCC RAS OSCC RAS RAS

0.820 1.505 2.145 0.685 2.965 3.650

Standard error 0.228 0.228 0.228 0.228 0.228 0.228

miRNA-145

P-value a

0.01538 0.00000b 0.00000b 0.06812c 0.00000b 0.00000b

Mean difference 0.07 0.06 0.46 0.13 0.39 0.52

Standard error 0.042 0.042 0.042 0.042 0.042 0.042

miRNA-184

P-value c

0.593 0.725c 0.00000b 0.054c 0.00000b 0.00000b

Mean difference 0.675 1.500 1.015 0.825 1.690 2.515

Standard error 0.155 0.155 0.155 0.155 0.155 0.155

P-value 0.00163a 0.00000b 0.00000b 0.00005b 0.00000b 0.00000b

P < 0.05 Significant. P < 0.001 Highly significant. c P > 0.05 Nonsignificant. a

b

able was that miRNA-184 was the only tested miRNA that registered a significant difference between OSCC and PMD with dysplasia. When a ROC curve was designed for each miRNA, the cutoff point delineating the occurrence of malignant change was a fourfold increase in miRNA-21 with specificity 65% and sensitivity 65%, or a 4.5-fold increase with specificity 90% and sensitivity 60% (Figure 2). On the other hand, miRNA-145 showed a cutoff point of a 0.6 decrease rate, with specificity 70% and sensitivity 60% (Figure 3). Regarding miRNA-184, the cutoff point was a threefold increase, with specificity 75% and sensitivity 80% (Figure 4). When AUC was calculated, the following results were obtained: the area under miRNA-21 ROC curve was 0.73, the area under miRNA-145 ROC curve was 0.68, and the area under miRNA-184 ROC curve was 0.86, denoting that the diagnostic accuracy of miRNA-184 is good and for miRNA-21 is fair, while for miRNA-145 is poor.

Discussion Oral potentially malignant disorders (PMDs) can progress from normal to mild dysplasia, moderate dysplasia, and severe dysplasia to carcinoma in situ and finally invasive OSCC. As clinical and histological assessments are not reliable in predicting which of these precursor lesions will progress (Scully, 2014), the search for genetic biomarkers of tumor progression is intense. Salivary microRNA (miRNA) expression has been proposed as a mean to identify signatures associated with

Figure 2 ROC for miRNA-21

cancer initiation and progression (Yang et al, 2013). miRNAs are small noncoding RNAs of 18–25 nucleotides in length with a crucial role in posttranscriptional regulation of gene expression. By base pairing to the 30 -untranslated region (30 -UTR) of target miRNAs, the mature miRNA is incorporated into the RNA-induced silencing complex (RISC) where it mediates gene expression by binding to target miRNA (Hannon and Rossi, 2004). In the present study, we investigated the expression of 3 miRNAs in the saliva of patients with mucosal lesions Oral Diseases

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ROC micro-RNA 145

1 0.9 0.8

Sensitivity

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0.7

0.8

0.9

1

Specificity

Figure 3 ROC for miRNA-145

ROC micro RNA-184

1 0.9 0.8

Sensitivity

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0

0.1

0.2

0.3

0.4

0.5

0.6

Specificity Figure 4 ROC for miRNA-184

—oral potentially malignant disorders (PMDs) and oral squamous cell carcinoma (OSCC), in comparison with healthy control subjects and, as disease controls, patients with one of the common oral inflammatory lesions—recurrent aphthous stomatitis (RAS). The inclusion of an inflammatory, immunologically mediated, ulcerative lesion as a positive control was to delineate the miRNA salivary level characteristic for dysplastic lesions—because the upregulation of miRNAs such as miRNA-21 has been reported in some inflammatory conditions (Raisch et al, 2013). The miRNAs included in the present investigation were miRNA-21 and miRNA-184, as these were previously reported to be overexpressed in tissue specimens and cell lines of OSCC (Kolokythas et al, 2011), as well as miRNA-145, with its known tumor suppressor effect (Zhang et al, 2013) to elucidate the usefulness of their salivary levels analysis in detecting PMD and lesions with actual malignant transformation. miRNA-21 is a key onco-miRNA that is upregulated in several malignancies (breast, pancreas, lung, gastric, Oral Diseases

prostate, colon), and head and neck cancers (Asangani et al, 2008; Yan et al, 2008; Hwang et al, 2010; Reis et al, 2010; Fu et al, 2011) many of which are OSCC. miRNA-21 is overexpressed in the proliferation, invasiveness, metastasis, and chemosensitivity of tumors (Asangani et al, 2008; Yan et al, 2008; Hwang et al, 2010; Reis et al, 2010; Zhang et al, 2012). The study by Kawakita et al (2013), utilizing in situ hybridization, was in accordance with the present results, where the level of miRNA-21 expression was significantly higher in OSCC tissues than in adjacent normal oral tissues. Their results showed that miRNA-21 overexpression might play a pivotal role in OSCC tumorigenesis and invasiveness through the Wnt/b-catenin pathway, by targeting DKK2. A systematic review and meta-analysis showed that miRNA-21 overexpression resulted in poor survival of patients with a variety of carcinomas (Fu et al, 2011). Other studies showed that several molecules, such as phosphatase and tensin homolog deleted on chromosome ten (PTEN) (Han et al, 2012b; Zhang et al, 2012; Liu et al, 2013), programmed cell death 4 (PDCD4) (Asangani et al, 2008; Reis et al, 2010), and matrix metalloproteinase inhibitor RECK (Zhang et al, 2012), were all targets for miRNA21, suggesting that miRNA-21 is indeed an important oncogenic miRNA closely related to tumor invasion. Han et al (2012a,b) demonstrated that miRNA-21 can regulate cell invasiveness of breast cancer via AKT (also known as ‘protein kinase B’, PKB) and ERK1/2 (extracellular signal-regulating kinase) pathways by targeting PTEN. Furthermore, Asangani et al (2008) reported that tumor suppressor PDCD4 (programmed cell death 4) could be negatively regulated by miRNA-21 at the posttranscriptional level via a specific target site within the 30 -UTR in colorectal cancer. In the present study, our demonstration of the highly significant increase in salivary level of miRNA-21 in OSCC when compared with all other groups, except that of dysplastic PMD lesions, highlights its value in delineating these lesions from inflammatory and nondysplastic conditions. Moreover, the lack of significant difference between OSCC and PMD showing dysplastic changes could suggest that the aberrant expression of miRNA-21 in saliva might reflect an early molecular event in the pathogenesis of OSCC. A 4- to 4.5-fold increase (specificity 65%, sensitivity 65% and specificity 90%, sensitivity 60%, respectively) seemed quite reasonable as a cutoff point for frank cancer transformation. miRNA-184 in the present study showed a highly significant increase in salivary level in OSCC when compared to any other group, with a reasonable cutoff point, threefold increase (75% specificity and 80% sensitivity). Also, it was the only studied miRNA that discriminated between OSCC and PMD with dysplasia, with a highly significant statistical difference. This is in accordance with previous studies that have reported increased levels of miRNA-184 in OSCC. Wong et al (2008) showed a 59fold higher miRNA-184 expression in OSCC cells compared with paired normal cells. MicroRNA-184 was identified to be among the 24 upregulated mature microRNAs by at least a threefold expression difference in laser microdissected cells from four OSCC and paired normal controls studied by these authors. It was postulated that

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miRNA-184 acts as an oncogene by inducing proliferation and inhibiting apoptosis potentially by targeting c-Myc. They also concluded that Inhibition of miRNA-184 in tongue SCC cell lines could reduce cell proliferation rate. However, another study (Yu et al, 2008) presented a different explanation for the effects of miRNA-184 on epithelial cells and cancer cell lines, including Cal27 (an aggressive cell line) via AKT signaling. Suppression of the AKT pathway that is associated with increased cell apoptosis and death was demonstrated with ectopic expression of miRNA-184. miRNA-145 results shown here are also in accordance with previous reports that showed miRNA-145 underexpression in most head and neck carcinoma samples, particularly a recent study by Yang et al (2013) who reported decreased salivary miRNA-145 in OSCC. Many studies have identified several targets for miRNA-145, including K-RAS (Chen et al, 2009), Fli1 (Zhang et al, 2011), cMyc (Sachdeva et al, 2009), and DFF45 (Zhang et al, 2010). These studies have also demonstrated that this miRNA suppressed tumors, decreased proliferation, or promoted apoptosis. Restoring miRNA-145 expression in OSCC cells dramatically suppressed cell proliferation and colony formation and induced G1 phase (or G1/S transition) arrest and cell apoptosis. Importantly, miRNA-145 downregulated the expression of c-Myc and Cdk6 (cyclindependent kinase 6) (Shao et al, 2013). In addition, miRNA-145 can negatively modulate the expression of MDM2 (Zhang et al, 2013), an oncogene, overexpressed in many human tumors, that enhances cellular transformation (Zhang et al, 2012) and a target for p53. miRNA-145 is posttranscriptionally activated by upregulated p53, thereby generating a short miRNAs-MDM2-p53 feedback loop. Re-expression of miRNAs suppresses cellular growth and triggers the apoptosis of epithelial tumors, in vitro and in vivo, by enhancing p53 activity via MDM2 turnover. Moreover, the miRNA-dependent MDM2 turnover contributes to the equilibrium of repeated p53 pulses in response to DNA damage stress. These findings suggest that MDM2 dysregulation caused by downregulation of miRNA-145 contributes to cancer development and has a key role in regulating cellular proliferation and apoptosis. As miRNA-145, both in vivo and in vitro, causes MDM2 downregulation and subsequent p53, p21, and BAX upregulation, some authors have postulated that attempting the re-expression of miRNA-145 may be a reasonable strategy for treatment of cancers (Zhang et al, 2013). Furthermore, Sachdeva and Mo (2010) have shown that miRNA-145-mediated suppression of cell invasion is, in part, due to the silencing of the metastasis gene mucin 1 (MUC1), and they found that ectopic expression of MUC1 (which enhances cell invasion) can be blocked by miRNA-145. Of interest, suppression of MUC1 by miRNA-145 causes a reduction of b-catenin which is a target of miRNA-21 (Kawakita et al, 2013) but in the reverse direction. Han et al (2012a,b) demonstrated that the bcatenin pathway, in turn, regulated miRNA-21 expression via STAT3—playing a role in promoting cell invasion and proliferation. Thus, it seems that the decreased expression of miRNA-145 leads to an increase in b-catenin (Gonzalez-Moles et al, 2014) and consequent increased

miRNA-21 expression. Conversely, miRNA-21 and miRNA-184 seem to act synergistically on some common pathways such as the AKT pathway (Yu et al, 2008; Han et al, 2012b), inducing cell proliferation and inhibiting apoptosis. As pointed out by Spielmann and Wong (2011), early detection is a key question that needs to be addressed in almost all types of diseases. This could be achieved by saliva diagnostics which are very attractive because of noninvasive sample collection and simple sample processing and easy accessibility as compared with tissue biopsies. Therefore, developing highly sensitive and accurate assays for salivary biomarkers makes saliva a valuable diagnostic fluid (Herr et al, 2007). However, single biomarker detection is not effective enough for accurate diagnosis and medical decisions because of the complexity of the human biologic system and the high possibility of false-positive and false-negative results. The combination of multiple biomarkers would be more efficient. However, to date, no technology has been reported addressing the multiplexing mode including the measurement of RNA, protein, and small molecules.

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Conclusion It might be possible to use miRNAs such as salivary miRNA-21, miRNA-145, and miRNA-184 as noninvasive, rapid diagnostic biomarkers for oral malignant transformation, with miRNA-184 being the most precise. However, due to the level of their specificity and sensitivity, for the time being, they could furnish a confirmatory or follow-up aid in conjunction with biopsy, which remains the gold standard. Other miRNAs have also been recently implicated in OSCC, such as miRNA-140, miRNA-155, and miRNA-146A (Kai et al, 2014; Palmieri et al, 2014; Shi et al, 2014), confirming the need for continuing studies in this area.

Conflict of interest The authors declare no conflict of interest.

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