Salivary exosomal miR-24-3p serves as a potential detective biomarker for oral squamous cell carcinoma screening

Biomedicine & Pharmacotherapy 121 (2020) 109553

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Original article

Salivary exosomal miR-24-3p serves as a potential detective biomarker for oral squamous cell carcinoma screening

T

Lihong Hea,b,1, Fan Pinga,b,1, Zhaona Fana,b, Chi Zhanga,b, Miao Denga,b, Bin Chenga,b,*, Juan Xiaa,b,* a b

Department of Oral Medicine, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, PR China Guangdong Provincial Key Laboratory of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Oral squamous cell carcinoma Salivary exosomes miR-24-3p Diagnostic biomarker PER1

Objectives: miRNAs in salivary exosomes are used as novel non-invasive biomarkers for detection strategies of human disease. Here, we aimed to investigate the diagnostic potential of salivary exosomal miRNAs as biomarkers for screening oral squamous cell carcinoma (OSCC) and to explore the underlying mechanisms of OSCC pathogenesis. Materials and methods: Differentially expressed miRNAs were obtained from salivary exosomes of four healthy controls and four OSCC patients using miRNA microarray analysis. The expression of miR-24-3p in the salivary exosomes was then verified by qRT-PCR. The diagnostic power was assessed by receiver operating characteristic (ROC) analysis. Cell proliferation was measured using CCK-8 cell viability assay and colony formation assay. The target gene of miR-24-3p was confirmed by dual luciferase reporter assay. Results: A total of 109 miRNAs were found to be more than 2-fold altered in the salivary of patients and healthy individuals by miRNA microarray. qRT-PCR analysis further confirmed a significant increase of miR-24-3p in the salivary exosomes from 45 preoperative OSCC patients compared to 10 normal controls. ROC analysis showed that miR-24-3p has excellent diagnostic accuracy for OSCC (area under the ROC curve [AUC] = 0.738; P = 0.02). Similarly, we found that miR-24-3p expressed a higher level in OSCC neoplastic tissues, suggesting that circulating miR-24-3p may originate from tumor cells. Furthermore, exogenous exosomal miR-24-3p increased proliferation of recipient malignant cells. Additionally, overexpression of miR-24-3p promoted the proliferation of OSCC cells and regulated the expression of cell cycle-related genes. Dual luciferase reporter assay indicated that miR-24-3p can interact with PER1 directly. Conclusions: Salivary exosomal miR-24-3p is a potential novel diagnostic biomarker for OSCC, and miR-24-3p can maintain the proliferation of OSCC cells through targeting PER1.

1. Introduction Oral squamous cell carcinoma (OSCC) is one of the most common cancer types in the world, accounting for about 90% of malignancy in the oral cavity. In 2018, there were about 354,864 new lip/oral cavity cancer cases and 177,384 deaths worldwide [1]. Although the oral lesions of OSCC are readily accessible for clinical observation, patients lost the best timing for diagnosis and treatment due to the unapparent symptoms in early stages. Despite improvements in surgical techniques and chemotherapy, the prognosis of OSCC is still pool, with a 5-year overall survival (OS) rate of only about 64.4% [2]. Therefore, it is an urgent demand to search specific and sensitive molecular biomarkers

for early detection, which can also provide potential therapeutic targets for OSCC. Exosomes are lipid bilayer-enclosed extracellular vesicles (EVs) of 50–150 nm in diameter containing various molecules such as proteins, mRNA, and microRNAs (miRNAs). Exosomes originate from multivesicular bodies (MVBs), which are formed in the endolysosomal pathway by inward budding of the endosomal membrane. They are released into extracellular space and enter the circulation by fusion of MVBs with the plasma membrane [3]. Exosomes secreted by cells or organs are existed in all body fluids, including blood, urine, saliva and so on. Thus, circulating exosomes have attracted much attention due to their potential role as novel tumor biomarkers for future clinical

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Corresponding authors at: No. 55 Linyuan Xi Road, Guangzhou, Guangdong 510055, PR China. E-mail addresses: [email protected] (B. Cheng), [email protected] (J. Xia). 1 Equal contribution. https://doi.org/10.1016/j.biopha.2019.109553 Received 5 July 2019; Received in revised form 9 October 2019; Accepted 9 October 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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applications [4]. Human saliva, as a readily available body fluid, may be a powerful indicator for evaluation of both local and systemic diseases. miRNAs are a type of single-stranded, non-coding short RNA molecules of approximately 20 nucleotides in length, which play important roles in the modulation of variety physiological and pathological processes[5]. Recent studies have demonstrated that miRNAs in salivary exosomes can be used as minimally invasive diagnostic biomarkers for different malignancies [6–9]. They are highly stable and have a long life span, as miRNAs in exosomes are protected from RNase in body fluids by encapsulated in the phospholipid bilayer membrane [10]. In the present study, we explored the potential role of miR-24-3p that contribute to the pathogenesis of OSCC and demonstrated that miR-24-3p in salivary exosomes might be a non-invasive molecular biomarker for the diagnosis of OSCC.

treatments such as surgical resection, chemotherapy and radiotherapy. 5 ml of unstimulated whole saliva was obtained from patients and health controls under standard conditions as previously described [11]. Briefly, all subjects were instructed not to eat, drink coffee or caffeinated soft drinks, chew gum or brush their teeth for at least one hour prior to saliva collection. Each participant was requested to rinse their mouth with clear water and then rest for 5 min. Subsequently, their saliva was collected through chilled 50-ml conical tubes from 9.00 a.m. to 12.00 p.m. within 30 min. After collection of saliva, it was immediately centrifuged at 2,600 g for 30 min at 4 °C to reduce contamination of cells debris, bacteria and any food residuals. The supernatants were separated and stored at −80 °C for further analysis. 2.2. Cell culture OSCC cell line HSC6 was generously provided by Professor J. Silvio Gutkind (National Institutes of Health, Bethesda, MD, USA). The SCC25 cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). HSC6 was cultured in DMEM (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS). SCC25 was cultured in DMEM/F12 (GIBCO, USA) with the same concentration of FBS. The cells were maintained at 37 °C in a humid cell culture incubator with 5% CO2.

2. Material and Methods 2.1. Study participants and saliva collection In all, saliva samples were obtained from 49 patients with OSCC at the Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Sun Yat-sen University. Besides, 14 healthy volunteers were recruited as normal controls. OSCC tissue and adjacent normal tissue specimens were collected from another 30 OSCC patients undergoing surgery. All subjects considered eligible for inclusion in this study must meet the following criteria: All OSCC patients were diagnosed for the first time by tissue biopsy and histopathologic examination. All the patients did not receive clinical interventions such as chemotherapy and radiation before surgery. Exclusion criteria for all participants included the presence of any other malignant tumors, oral mucosal lesions and severe systemic diseases. Additionally, there was no significant difference in the distribution of age and gender between OSCC patients and healthy controls (P > 0.05). To exclude the impact of risk factors on miRNA differential expression analysis between OSCC group and control group, basic information on history of smoking and alcohol consumption were collected. Clinical parameters of the OSCC group and normal group are depicted in Table 1. The data represented that there was no significant differences in history of smoking and drinking between two groups (P > 0.05).The Hospital of Stomatology, Sun Yat-sen University Ethics Committee ratified our study, and written informed consents were acquired from all participants before the experiments. Saliva collection of OSCC patients was carried out prior to cancer

2.3. Exosome preparation Exosomes were extracted from saliva specimens (0.5–1.0 ml) and supernatants of OSCC cells (cultured in exosome-free medium) using ExoQuick-TCTM (SBI, Mountain View, CA, USA). Briefly, cell-free saliva and cleared cell culture medium was fully incubated with total exosome isolation reagent overnight at 4 °C, followed by centrifugation at 1500 g for 30 min at 4 °C to precipitate the exosomes. Transmission electron microscopic (TEM) analysis was used for morphology examination of isolated vesicles. Typical negative staining was performed on exosomes, and then exosomes were observed and photographed by JEM1400 electron microscope (JEOL, Japan) as described previously [12]. The size (nm) distribution, mean size and concentration (particles/ml) of the exosomes were detected by Nanoparticle Tracking Analysis (NTA) using a NanoSight NS300 instrument. 2.4. Quantitative RT-PCR TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from exosomes, cells and tissues. Specifically, 25 fmol of synthetic Cel-miR-39 mimic was mixed with each exosomes specimen as an internal control after the lysis step [13]. Total RNA (500 ng) was reverse-transcribed into cDNA using PrimeScript™ RT Reagent Kit (Takara Biotechnology Co., Ltd, China). The qRT-PCR analysis was performed using SYBR Green Master Mix (Roche) on the LightCycler® 96 System machine (Roche Diagnostics GmbH, Mannheim, Germany).The amplification protocol included an initial denaturation step at 95 °C (10 min), followed by 45 cycles at 95 °C (15 s) and then 60 °C (1 min), and 72 °C (15 s). The Cel-miR-39, U6 and GADPH were acted as internal controls. The relative gene expression values were analyzed using the ΔΔCt method.

Table 1 Clinical parameters of the OSCC patients and healthy subjects. P-valuea

Tissue sample (N = 30)

Saliva sample from Patients (N = 49)

Saliva sample from healthy controls (N = 14)

Age (n, %) ≤50 years > 50 years

14(46.7) 16(53.3)

22(44.9) 27(55.1)

7(50.0) 7(50.0)

0.736

Gender (n, %) Male Female

18(60.0) 12(40.0)

30(61.2) 19(38.8)

8(57.1) 6(42.9)

0.783

Tobacco smoking (n, %) Yes 13(43.3) No 17(56.7)

20(40.8) 29(59.2)

5(35.7) 9(64.3)

0.731

Alcohol consumption (n, %) Yes 12(40.0) No 18(60.0)

13(26.5) 36(73.5)

3(21.4) 11(78.6)

0.699

Characteristic

2.5. miRNA microarray analysis In all, eight exosomal RNA samples (4 from OSCC patients and 4 from normal individuals) were selected and used for microarray analysis. The intensity of each hybridization signal was detected using the Human miRNA Microarray platform (8 x 60 K, Release 21.0; Agilent Technologies, Santa Clara, CA, USA) and Microarray Scanner (Agilent Technologies), which encompassed 2568 human miRNAs. Image processing was performed using the Feature Extraction software. The generated miRNA profile was normalized to the amount of input total

a Statistical analysis was performed to compare differences in clinical characteristics between two groups of saliva sample.

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RNA, and the P value was adjusted by the Benjamini-Hochberg algorithm to control the false discovery rate. After normalizing the data using GeneSpring GX software, the gene expression differences the statistically significant p-value can be calculated, and a difference fold change (Log FC) between the two sets of samples is obtained. The unsupervised hierarchical clustering analysis of differentially expressed miRNAs was performed using the clustering method in R language and its extension package.

in triplicate, and the quantification of the band intensity was performed using Image J software. 2.10. Cell proliferation Cell viability was detected using the cell counting kit-8(CCK-8) assay kit (Dojindo, Gaithersburg, USA). The OSCC cells (2.0–3.0 × 103/ well) were seeded into 96-well plates and treated with 10 μg/mL exosomes (quantified by BCA Protein Assay), miR-24-3p expression vector, control vector for 24 h. Premixed CCK-8 and medium(10 μl:90 μl) were added into per well and then incubated for 0.5–2 h at 37 °C. Absorbance was assessed by a spectrophotometer (Bio-Tek, Elx800, USA) at 450 nm.

2.6. Prediction and preliminary validation of target gene of miR-24-3p The potential target genes of miR-24-3p (MIMAT0000080) were predicted by TargetScan v.7.2 (http://www.targetscan.org/vert_72/), miRDB (http://mirdb.org), and miRWalk v.3.0 (http://mirwalk.umm. uni-heidelberg.de/) online databases. In order to improve the reliability of bioinformatics tools, overlapping genes between the above target genes were considered to be reliable putative target genes of miR-243p. Luciferase reporter assays were carried out to verify that miR-24-3p directly interacted with the 3′-UTR of PER1. Luciferase reporter plasmids containing the wild-type 3′-UTR of PER1 (PER1-3′UTR-WT) or the 3′-UTR mutant form of PER1 (PER1-3′UTR-MUT) were purchased from OBiO Technology (Guangzhou, China). 50 nmol miR-24-3p mimics or control mimics were co-transfected with luciferase reporter vectors (500 ng) and 0.01 mg Renilla luciferase plasmid into HSC6 and SCC25 cells using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA). Following an incubation of 48 hours, the firefly and Renilla luciferase activity of the cell lysates were detected by the Dual-Luciferase Reporter Assay Kit (Promega Corporation, Madison, WI, USA).

2.11. Colony formation assay The transfected HSC6 and SCC25 cells were seeded into 6-well plates at a density of 500 cells per well. After approximately two weeks of culture, the medium was discarded, the colonies were fixed and stained with 0.5% crystal violet for 30 minutes and then imaged with a digital camera to record the results. 2.12. Statistical analysis We used SPSS version 19.0. software (SPSS, Inc. Chicago, IL) and GraphPad prism 5.0 software to perform statistical analyses. The results are presented as mean ± standard deviation (SD) or SEM (where indicated) with a Student's t-test. All experiments were performed in triplicate. Differences in clinical characteristics between the two groups of saliva samples were compared using the Pearson’s chi-squared test. The receiver operating characteristic (ROC) curve analysis was applied to obtain the area under the ROC curve (AUC) to evaluate the potential diagnostic accuracy of miR-24-3p for OSCC [14]. The optimal cutoff threshold value for diagnosis was selected by Youden index (sensitivity + specificity − 1). P < 0.05 was taken to indicate a statistically significant.

2.7. TCGA data preparation and integration The original data of RNA-seq for 372 samples (340 OSCC and 32 normal samples) was obtained from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/repository). The Edge R package was applied to acquire the mRNA expression matrix. Subsequently, the expression level of PER1 in each case was calculated and normalized by log2 transformed.

3. Results 3.1. Candidate exosomal miRNA biomarker for OSCC

2.8. Cell transfection We isolated salivary exosomes from healthy controls and OSCC patients. Western blotting was performed to verify the molecular characterization of salivary exosomes. The specific exosomal markers (CD63, CD81, TSG101) were showed in both samples (Fig. 1A). The images of salivary exosomes under transmission electron microscopy revealed the typical round-shaped vesicles surrounded by bilayer lipid membranes with diameters ranging of 50–150 nm (Fig. 1B). Nanoparticle-tracking analysis (NTA) of exosomes isolated from OSCC patients and healthy donors indicated that most of the particles fell within the expected size range of 50–150 nm in diameter with the highest peak around 110 nm. The Particle concentrations of exosomes from patients and healthy controls were 8.43 × 107and 4.32 × 108 per mL of saliva, respectively (Fig. 1C, D). Their molecular and morphological features verified that the pellets extracted from the salivary samples were exosomes. Exosomal RNA samples were obtained from four healthy controls and four patients were performed for miRNA microarray analysis. As shown in Fig. 1E and F, the miRNA expression profiling of salivary exosomes differed among OSCC patients and healthy controls. After normalization of the microarray data, differentially expressed genes were screened with a 2- or 0.5-fold change (FC) cut-off value and a P ≤ 0.05 compared to the normal group. The results showed that in comparison with the healthy group, there were 109 miRNAs exhibited changes in their expression levels, of which 50 miRNAs significantly increased (fold change 3.60 to 345.62) and 59 miRNAs decreased (fold change 0.02 to 0.49) (Table S1). The hierarchical clustering analysis

The miR-24-3p expression vector (miRNA mimic), control vector (scrambled negative control) were produced by Ribobio Life Technologies (Guangzhou, China). The Lentiviral plasmids encoding miR-24-3p and sponge-miR-24-3p, or negative controls were purchased from Cyagen Biosciences (Guangzhou, China). Transfections of miR-243p were conducted using Lipofectamine 3000 (Invitrogen). 2.9. Western blotting Protein was lysed with 100 μl ice-cold 1X RIPA buffer (Beyotime) supplemented with 1:100 protease inhibitors (Thermo Scientific) and centrifuged for 20 min (12,000 rpm, 4 °C). Protein concentration was determined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Approximately 60 μg total protein extracts were separated on 10–12% SDS-PAGE and then transferred onto to a polyvinylidene fluoride membrane (Millipore, USA). Subsequently, the membranes were blocked out and then incubated with specific primary antibodies overnight at 4 °C, including those against CD81, CD63, TSG101, P21, P27, CDK2, CDK4, CDK6, CyclinD1, CyclinE, CyclinB1, and GAPDH (Abcam and Cell Signaling Technology, 1:1000). The membranes were washed with TBST (PBS-Tween20), and then incubated with streptavidin-horseradish peroxidase (HRP) secondary antibodies at room temperature for 1 h followed by washing. Chemiluminescence measurement was performed by ECL-advance Western Blot Detection System (Bio-Rad, California, USA). Experiments were performed at least 3

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Fig. 1. The differential expression of salivary exosomal miRNAs between healthy controls and patients with OSCC. (A) Western blotting analysis of the exosomal markers (CD63, CD81, TSG101). (B) Transmission electron microscopy of saliva-derived exosomes showing roundshaped vesicles of different sizes. (C, D) The size distribution and concentration of exosomes isolated from OSCC patients and healthy donors. Data are presented as mean ± SD. (E) The heat map and hierarchical clustering diagram representing differentially expressed miRNAs of salivary exosomes in OSCC patients and healthy individuals. Expression levels are represented by colored gradation, yellow color represents high expression level, and blue color represents low expression level. (F) Scatter plot showing the differential miRNA expression profiles between patients and healthy controls derived exosomes. Red dots indicate upregulated miRNAs; green dots indicate downregulated miRNAs; black dots indicate miRNAs with no difference in expression level.

not been studied in any human malignancy or other disease [15–18]. Moreover, Alles et al. failed to confirm that miR-7975 is a true miRNA in human cell lines by northern blotting (NB), which is an essential quality criterion for true miRNAs [19]. miR-1246 contained in circulating exosomes has been well-evaluated as biomarkers for early detection of pancreatic cancer [20], breast cancer [21], prostate cancer [22], hepatocellular carcinoma[23]. Notably, miR-24-3p has been

which was performed based on the normalized microarray expression data of differentially expressed miRNAs could successfully separate the healthy controls from the patients with OSCC. Among the differentially expressed miRNAs, the top three upregulated miRNAs were miR-7975, miR-1246 and miR-24-3p, which showed the largest differences. However, in addition to verifying its expression in miRNA microarray analysis, the function of miR-7975 has 4

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exosomes secreted by OSCC cells could affect the biological function of recipient malignant cells. The overexpressing and knockdown lentiviral plasmids of miR-24-3p were used to induce stable up-regulation and down-regulation of miR-24-3p in OSCC cell line HSC6 and SCC25 (Fig. S1A). Next, OSCC cells were co-cultured with exosomes derived from those stable cell lines. qRT-PCR analysis confirmed the overexpression of miR-24-3p in lentivirus-miR-24-3p OSCC cell-derived exosomes, and a reduction in miR-24-3p-sponge exosomes (Fig. 3C). The results of CCK-8 assays showed that the cell viability was increased under the stimulation of miR-24-3p overexpression OSCC cell-derived exosomes, whereas miR-24-3p-sponge exosomes showed opposite effects (Fig. 3D). Collectively, these findings revealed that OSCC cells overexpressing miR-24-3p in the tumor microenvironment may further secrete exosomal-miR-24-3p to produce a ‘tumor-friendly’ environment that promotes tumor progression.

reported to be up-regulated in head and neck squamous cell carcinoma (HNSCC) tissues and acts as an oncogene in HNSCC [24]. However, few studies have been carried out to evaluate the diagnostic value of miR24-3p contained in circulating exosomes for human malignancies. Based on the microarray analysis, the expression level of miR-24-3p (fold change 121.54) in salivary exosomes from OSCC was increased significantly compared with healthy controls, ranking third among the most altered miRNAs. Therefore, in order to explore more specific OSCC biomarker, we selected miR-24-3p, a highly reliable human miRNA, for subsequent studies. 3.2. Levels of salivary exosomal miR-24-3p discriminate OSCC patients from healthy controls To determine the specificity of altered expression levels of salivary exosomal miR-24-3p for OSCC, we next examined the level of exosomal miR-24-3p in saliva samples from 45 OSCC patients and 10 healthy controls by qRT-PCR. The synthetic C. elegans Cel-miR-39 spike-in was used as a reference control to correct for differences caused by exogenous factors during miRNA extraction process. The results showed that the mean ± SE value of relative expression of miR-24-3p was 1.769 ± 0.5719 for the normal specimens, and the mean ± SE value of the cancer group was 10.13 ± 1.961. The level of salivary exosomal miR-24-3p was 5.73-fold higher in OSCC patients than in controls (P < 0.01) (Fig. 2A). To determine whether the level of salivary exosomal miR-24-3p can be used to distinguish patients with OSCC from healthy controls, we constructed ROC curve to evaluate the discrimination power of miR-243p for OSCC. Comparing OSCC subjects with controls, AUC of miR-243p was found to be 0.738 (95% CI: 0.589–0.886, P = 0.02).The sensitivity and the specificity of miR-24-3p were 64.4% and 80% (Fig. 2B). The results demonstrate that the exosomal miR-24-3p in salivary has great potential as a diagnostic biomarker for OSCC.

3.4. miR-24-3p promotes proliferation of OSCC cells in vitro To further investigate the molecular mechanisms of miR-24-3p in tumor progression, we up-regulated the expression of miR-24-3p in OSCC cells by transfection of miR-24-3p mimics (Fig. S1B). Results of CCK8 and colony formation assays showed that overexpression of miR24-3p significantly increased growth rate and colony formation efficiency of OSCC cells (Fig. 4A, B). To delineate the potential regulatory mechanism of miR-24-3p on cell proliferation, cell cycle-related genes were measured by Western blotting. The protein expression levels of Cyclin B1, Cyclin D1, Cyclin E1, CDK2, CDK4, and CDK6 were significantly higher in the miRNA-243p mimic group than in the control group, each of which promotes cell proliferation. Besides, the protein expression levels of P21 and P27 were significantly lower in the miRNA-24-3p mimic group than in the control group (Fig. 4C). The expression levels of protein have been calculated and presented quantitatively in Figure S2.

3.3. OSCC tissues and cells secrete exosomes containing miR-24-3p

3.5. PER1 is a direct target of miR-24-3p

Since the expression of exosomal miR-24-3p is up-regulated in saliva from OSCC patients, the expression levels of the miR-24-3p were examined in additional 30 pairs of OSCC and corresponding normal tissues to validate the consistency of the miRNAs in saliva and tissue of OSCC patients. Relative expression of miR-24-3p in OSCC neoplastic and peritumoral tissues was measured by qRT-PCR. Compared with peritumoral tissues, the expression of miR-24-3p in neoplastic tissues significantly increased in 15 of 30 pairs of specimens, decreased in seven pairs of specimens and there was no significant difference in eight pairs (*P < 0.05; **P < 0.01) (Fig. 3A). As shown in Fig. 3B, the relative expression of miR-24-3p was significantly (P < 0.05) higher in OSCC tissue samples than in their matched non-tumor tissues, which maintained the consistency in salivary. Considering the important role of circulating exosomes on cell-tocell communication, we next assessed whether miRNA-containing

We predicted target genes of miR-24-3p using bioinformatics tools TargetScan7.2, miRDB and miRWalk (Fig. 5A). The list of overlapping candidate genes ranked by cumulative weighted scores for the three bioinformatics tools is provided in Supplementary Table 2. Among the candidate genes, Period 1 (PER1), which has been considered to be associated with proliferation and cell cycle progression of OSCC [25,26], was chosen for further study. As an important circadian clock gene, PER1 exhibits anti-proliferative effects in human cancer cells by modulating many key downstream cyclins to block cell cycle progression [27,28]. Differential expression analysis of PER1 in OSCC patients was conducted by collecting and processing data from the TCGA database. It was revealed that PER1 expression level was significantly decreased in OSCC compared to normal controls (P < 0.01) (Fig. 5B). Fig. 5C shows the predicted binding region between miR-24-3p and the 3′-UTR of PER1. The luciferase reporter assays were conducted to verify Fig. 2. Validation of the diagnostic power of miR-24-3p for OSCC. (A) qRT-PCR confirmation of elevated expression of the miR24-3p in salivary exosomes of OSCC patients compared to healthy controls. The data were normalized to Cel-miR-39 snRNA and represented as the mean ± standard deviation. **P < 0.01. (B) Receiver operating characteristic curve analysis of salivary exosomal miR-24-3p in discriminating between patients and healthy individuals. The diagnostic value of miR-24-3p expression for OSCC was statistically significant (P = 0.02). The black circle indicates optimal cutoff threshold value for diagnosis.

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Fig. 3. miR-24-3p was up-regulated in OSCC neoplastic tissues, and miR-24-3p delivered by exosomes promote proliferation of recipient OSCC cells. (A, B) Relative expression of miR-24-3p in OSCC neoplastic and peritumoral tissues by qRT-PCR. The bars correspond to the mean ± standard deviation, *P < 0.05, **P < 0.01. (C) qRT-PCR analysis of miR-24-3p expression in exosomes derived from OSCC cells transfected with the lentiviral plasmid containing miR-24-3p (miR-24-3p, miR-24-3p-sponge) or with the miR-control. (D) CCK-8 assay was used to evaluate the effect of exosomes on the cell viability of HSC6 and SCC25 cells. Data are presented as the mean ± SEM, *P < 0.05, **P < 0.01.

scientists since the method of sample collection for disease is cost-effective, accurate and noninvasive. Many studies have shown that DNA, RNA, proteins and metabolites contained in saliva can be effective indicators for both local and systemic cancers [29]. Diagnostic methods and appropriate biomarkers at the early stages of cancers can significantly improve patient outcomes and be used to predict and monitor responses to therapy. Furthermore, although saliva is directly exposed to environmental impacts, the quality and expression levels of RNA and DNA obtained from saliva were comparable to those in other body fluids or cellular samples [30,31]. miRNA, which play a crucial role in modulating various pathogenic processes of cancers through their interaction with target mRNA, has been widely described in salivary. Salivary miRNAs have demonstrated to be potential biomarkers for various diseases including oral cancers [32–36]. Exosomal microRNAs exhibits attractive advantage for cancer diagnosis and prognosis, since the lipid bilayer of exosomes can protect miRNA from degradation by RNase in body fluids [10]. Moreover, Gallo et al compared the miRNA between the exosomal pellets and the exosome-depleted saliva. It is revealed that the majority of miRNAs are concentrated in exosomes [37], which facilitating signal amplification

the hypothesis that miR-24-3p could directly target PER1. The results revealed that cells transfected with the miR-24-3p mimics showed a significantly decrease in luciferase activity of PER1 3′-UTR wild-type reporter, while the mutant-PER1 group showed no significant alteration in luciferase activity (Fig. 5D). In addition, we found that the mRNA expression levels of PER1 were significantly decreased in HSC6 and SCC25 cells transfected with miR-24-3p mimics compared to the miRNC transfected OSCC cells (Fig. 5E). When HSC6 and SCC25 were cultured with miR-24-3p overexpression OSCC cell-derived exosomes, the expression level of PER1 was decreased compared with the control group (Fig. 5F). Taken together, these data demonstrated that miR-243p could inhibit PER1 expression through directly targeting its 3′-UTR. 4. Discussion Traditional diagnostics for malignant tumors such as tissue biopsy and repetitive blood sampling can often be time-consuming and physically intrusive, adding excessive stress and pain to patients, thereby leading to poor patient compliance. In the last few decades, salivary diagnostics has attracted significant attention among clinicians and 6

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Fig. 4. miR-24-3p promotes proliferation of OSCC cells in vitro. (A) Growth curves of OSCC cells transfected with control or miR-24-3p mimic. Data are presented as the mean ± SEM, *P < 0.05, **P < 0.01. (B) Colony formation efficiency of OSCC cells transfected with control or miR-24-3p mimic. mean ± SEM. (C) The expression of cell cycle-related genes in OSCC cells.

(64.4% sensitivity and 80% specificity). Meanwhile, we identified that miR-24-3p was significantly elevated in neoplastic tissues compared to peritumoral tissues. The fairly consistent result from salivary exosomal miRNA-24-3p characterization to alterations in miR-24-3p expression in OSCC tumor tissues provide some evidence for the concept that OSCC exosomal microRNAs are released from the primary tumor site and can detectable in the circulation. In addition, in order to demonstrate the clinical diagnostic credibility of salivary exosomal miR-24-3p for OSCC, more evidence is needed for comparative studies between exosome derived from OSCC cells, serum and saliva. Notably, previous studies have shown that miR-24-3p is overexpressed in head and neck squamous cell carcinoma and can exert oncogenic functions [24,43,44]. However, the specific diagnostic role of miR-24-3p for OSCC is controversial, as abnormal expression of miR-24-3p is also found in several other kinds of cancers. Le et al. has demonstrated that miR-24 was highly expressed in the serum of patients with lung cancer [45], and miR-24-3p contributed to cell proliferation and motility of lung cancer via binding with SOX7 [46]. In breast cancer, level of miR-24-3p was found to be increased in plasma and tumor tissues of patients and was associated with tamoxifen resistance by directly targeting Bim [47,48]. miR-24-3p was also found to promote tumor progression in bladder cancer [49], hepatocellular carcinoma [50], and Hodgkin's lymphoma [51]. These previous reports are consistent with our current findings, suggesting that alteration of miR-24-3p expression may be a common characteristic of cancer patients, and that miR-24-3p may act as an oncomiR in various malignancies. The periodic circadian rhythm adjustment factor (PER1), a core circadian gene, plays an important role in the molecular mechanism of the circadian clock [52,53]. In addition, it can also exhibit tumor suppressor properties by participating in numerous signaling pathways such as cell proliferation, apoptosis, cell cycle progression and DNA damage response [27,28,54–56]. Recent reports have indicated low expression levels of PER1 in OSCC tissues, and targeted ablation of PER1 promotes malignant progression of OSCC by modulating the

of salivary miRNA and ensure high diagnostic sensitivity of human biologic fluids. However, the use of salivary exosomal miRNAs as biomarkers for human disease remains controversial. A critical limitation of using salivary exsomes for cancer screening is that differences in isolation techniques may alter the composition of purified subpopulations and the purity of the exosome pellets. In our study, we choose Total Exosome Isolation Reagent for isolating exosomes from saliva. This approach is a quick, simple and effective purification technique of exosomes and is therefore more feasible in clinical applications for cancer screening. However, the obtained exosomes pellets contain larger exosomes as well as higher levels of non-vesicular origin proteins in comparison to that achieved by classical differential ultracentrifugation [38,39]. Therefore, more efforts are to be made in standardizing and developing techniques to rule out the limitations of exosome isolation methods. It is worth mentioning that Peacock et al. reported that extracellular vesicle microRNA cargo is associated with HPV status in oropharyngeal cancer [40]. In addition, Takahashi et al. have shown that repeated smoking can alter the plasma miRNA profile of healthy individuals [41]. The affection of alcohol consumption in circulating exosomes has also been previously shown [42]. In this study, the authors found that the expression level of miRNA-122 was elevated significantly in sera exosomes of healthy individuals after alcohol binge drinking. These studies remind us that the composition of salivary exosomal miRNAs in healthy subjects may vary with external risk factors such as HPV infection, alcohol consumption, cigarette smoking, etc. Therefore, when salivary exosome miRNAs were used as biomarkers for disease, the diagnostic bias caused by the patients' exposure to the above-mentioned risk factors should be considered. In this study, we identified miR-24-3p as a candidate screening biomarker for OSCC due to its significantly higher expression in salivary exosomes from OSCC patients compared to those from normal individuals. Furthermore, in the ROC curve analysis, the AUC of miR-243p was 0.738, indicating that exosomal miR-24-3p could significantly distinguish OSCC patients from normal individuals with high accuracy 7

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Fig. 5. miR-24-3p directly target the 3′-UTR of PER1 and regulates its expression level in OSCC cells. (A) The target genes of miR-24-3p were predicted using bioinformatics tools TargetScan7.2, miRDB and miRWalk. (B) The Cancer Genome Atlas (TCGA) colorectal cancer database showed the expression levels of circadian rhythm-related gene PER1 in normal and OSCC group. (C) The putative binding site of miR-24-3p in the 3′UTR of wild-type PER1 predicted by databases is shown. (D) Luciferase reporter assays. HSC6 and SCC25 cells were co-transfected with reporters containing the wildtype or mutant form of 3′-UTR of PER1 mRNA and control miRNA or miR-24-3p mimic. (E) The PER1 mRNA expression levels of OSCC cells transfected with control, miR-24-3p mimic were determined by quantitative real-time PCR. (F) Relative expression of PER1 in HSC6 and SCC25 cells treated with miR-24-3p overexpression OSCC cell-derived exosomes. Data are presented as the means ± SEM, *P < 0.05, **P < 0.01. 8

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Fig. 6. A schematic diagram of proposed mechanism. Salivary exosomal miR-24-3p is a potential novel diagnostic biomarker for OSCC. Exosomal miR-24-3p derived from OSCC cells promotes proliferation of recipient malignant cells.

online version, at doi:https://doi.org/10.1016/j.biopha.2019.109553.

cyclin–CDK–CKI network [25,26]. We demonstrated that after increasing the expression of miR-24-3p in OSCC cells, the expression level of cyclin-dependent kinase inhibitors (CKI) P27 and P21 were significantly decreased, while that of cyclin family members (Cyclin B1, Cyclin D1, Cyclin E1) and cyclin-dependent kinase(CDK) family members(CDK2, CDK4, CDK6) were significantly increased. Thus our study provides valuable information for the role of miR-24-3p in the regulation of OSCC cells proliferation by targeting PER1. Interestingly, we found that exosomes containing miR-24-3p derived from transfected OSCC cells enhanced the proliferative capacity of the recipient OSCC cells, whereas inhibition of miR-24-3p attenuated this effect. Exosomes derived from cancer cells have been reported to have a tumor-promoting effect associated with the transfer of oncogenes [57–59]. However, exosomes are functional ensembles and malignant phenotypes may require a combination of miRNAs, mRNAs and functional proteins for gene system regulation. Therefore, determining the contribution of each component will be challenging. Further work is needed to understand whether and how each component of these tumor cell-derived exosomes contributes to a more aggressive phenotype of tumor cells. In conclusion, our study elucidated that increased expression of salivary exosomal miR-24-3p could reflect the cancer-bearing status of OSCC patients and that miR-24-3p is involved in regulating OSCC cell proliferation (Fig. 6). Therefore, miR-24-3p may serve as a clinical noninvasive salivary diagnostic biomarker and a novel therapeutic target for OSCC.

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Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (No. 81671000, 81870769) and the Science and Technology Program of Guangzhou, China (No. 201803010019, 201704020063). Appendix A. Supplementary data Supplementary material related to this article can be found, in the 9

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