Previous studies identified that the miR-31 expression level in oral potential malignant disorder (OPMD) is higher than that in normal oral mucosa, which is correlated with higher expression of vascular endothelial growth factor and lower expression of E-cadherin in OPMD. miR-31 expression was further upregulated in patients with recurrent OPMD and malignant transformation (37). Another previous study suggested that aberrant expression of miR-200c was associated with oral submucous fibrosis (OSF). The overexpression of miR-200c inhibited collagen gel contraction and migration and invasion of fibrotic buccal mucosal fibroblasts induced by arecoline via inhibition of zinc finger E-box binding homeobox 1 (ZEB1). Additionally, reverse transcription-quantitative PCR (RT-qPCR) analysis revealed that miR-200c was downregulated in 25 OSF samples compared with 25 normal mucosae (38). Brito et al (39) concluded that higher expression of miR-21 in OL was associated with increased mitotic figures, incremental nuclear/cytoplasmic ratio and hyperchromasia. Nylander et al (40) found that miR-21 was upregulated in 30 patients diagnosed with multifocal OLP compared with 10 healthy subjects, and in agreement, another study demonstrated that upregulation of miR-21 served a tumor-promoting role in oral cancer and upregulation of miR-21 was observed in 60 of 79 individuals with the disease (41). Aghbari et al (26) identified that miR-27b and miR-137 levels were downregulated in tissue and saliva samples of patients with OLP compared with those in normal controls. Among OLP subgroups, it was demonstrated that miR-137 exhibited the lowest expression level in the erosive type, suggesting that it serve as a biomarker for monitoring potential malignant transformation. The level of miR-375 in progressive lesions was significantly downregulated compared with that in non-progressive control lesions, and miR-375 expression was significantly downregulated in tissues following the transformation of premalignant lesions (including verrucous hyperplasia and verrucopapillary hyperkeratosis) into carcinoma, by comparison of premalignant lesions and oral carcinoma in situ (27). While only 2–3 and 0.4–2% of patients with OL and OLP, respectively, exhibit malignant transformation (42,43), further investigation of the roles of miR-21, miR-27b, miR-137, miR-200c and miR-375 may provide novel insights into pathways involved in the development of oral cancer.

Dysregulation of specific miRNAs has been previously reported in oral cancer. Wang et al (31) reported that miR-195-5p was significantly downregulated in 40 oral cancerous tissues compared with non-tumor tissues. Another study reported a distinct downregulation of miR-375 in 44 cancerous tissues compared with that in normal mucosae (32). Furthermore, a previous study revealed that miR-143 was downregulated in cancerous tissues compared with that in corresponding adjacent non-cancerous tissues in 81.6% (40/49) of patients (33). miR-802 was downregulated in 60.0% (12/20) of tongue squamous cell carcinoma (TSCC) cases compared with that in normal tissues (44). The expression levels of miR-137 (n=25) and miR-204-5p (n=52) were downregulated in oral cancer samples compared with those in matched normal tissues (45,46). Moreover, upregulation of specific miRNAs was observed in oral cancer tissues. Upregulation of miR-183 was identified in 68.3% (41/60) of TSCC tissues compared with adjacent non-cancerous tissues (47). The miR-373-3p expression level in oral cancerous tissues (n=63) was increased compared with that in adjacent non-cancerous tissues (48). The miR-155 expression level was upregulated in oral squamous cell carcinoma (OSCC) tissues (n=46) compared with that in normal oral mucosa, and the expression level was increased with increasing Tumor-Node-Metastasis (TNM) stage (49). miR-31, miR-182, miR-200a and miR-141 were significantly upregulated in cancerous tissues in 10 patients compared with adjacent non-cancerous tissues (50). The expression of miR-24 was significantly increased in TSCC tissues of 84 patients compared with adjacent non-cancerous tissues (51). Liu et al (52) demonstrated that 67% (10/15) of patients with primary oral cancer had increased miR-1275 expression in tumor tissues compared with that in adjacent tissues.

Previous studies showed that RNA in saliva is protected from degradation by binding to macromolecules such as apoptotic bodies and RISC, a mechanism also observed in plasma and serum RNAs (53–55). Park et al (56) analyzed saliva by immunoblotting analysis using an antibody against Ago2, and demonstrated that Ago2 was present in saliva, where it may confer stability to miRNAs. Furthermore, Park et al (56) found lower levels of miR-125a and miR-200a in the saliva of patients with oral cancer (n=12) compared with those in healthy controls (n=12), suggesting that the aforementioned miRNAs may serve as stable biomarkers of the disease. Liu et al (28) reported that the level of miR-31 in the saliva of patients with OSCC (n=45) prior to surgery was significantly increased compared with that in healthy subjects (n=24). Moreover, the miR-31 level in saliva samples was higher compared with that in plasma samples. The upregulation of miR-31 was detected with high sensitivity even in very small tumors, and the ability to detect miR-31 levels in the saliva of patients with small tumors was not significantly different compared with patients with advanced tumors, suggesting that salivary miR-31 may be utilized to detect and diagnose oral cancer lesions in high-risk populations. Zahran et al (29) reported a significant upregulation in salivary miR-21 and miR-184 levels in patients with oral cancer compared with those in healthy controls. Specifically, a four-fold increase in miR-21, with 65% specificity and 65% sensitivity, and a three-fold increase in miR-184, with 75% specificity and 80% sensitivity, were observed. The expression of salivary miR-145 was significantly decreased in patients with OSCC compared with clinically healthy controls, with 70% specificity and 60% sensitivity. Ries et al (30) reported that miR-3651 and miR-494 levels were upregulated, while the miR-186 level was significantly downregulated in whole blood samples of patients with recurrent tumors compared with non-recurrent controls. Therefore, miRNAs may be promising candidates for the development of diagnostic tools for oral cancer.

Certain miRNAs are known to be downregulated in tumor tissues compared with noncancerous tissues. Lower levels of miR-195-5p were associated with higher pathological differentiation grade (31). In comparison with patients without lymph node metastases (n=26), patients with lymph node metastases (n=18) had significantly downregulated miR-375. Furthermore, the overall survival of patients with oral cancer with low miR-375 expression (n=19) was lower than that of patients with high miR-375 expression (n=25) (32). Cao et al (34) revealed that advanced clinical stage and large tumor size of oral cancer were associated with low miR-26b expression. The 5-year survival rates of patients with low and high miR-26b levels were 26.7 and 53.3%, respectively.

On the other hand, certain miRNAs are upregulated in tumor tissues compared with adjacent non-cancerous tissues. Upregulation of miR-183 in patients with oral cancer markedly shortened the overall survival time and increased the risk of poor prognosis by 5.666 times. Upregulation of miR-21 resulted in a higher risk of short survival time (47). The expression of miR-373-3p was higher in primary tumors with metastases compared with that in tumors with no metastases (48). The upregulation of miR-24 was associated with advanced clinical stage in patients with oral cancer (51). A positive correlation between a high miR-155-5p level and cervical lymphatic metastases was observed, and the survival analysis of carcinomatous recurrence and metastasis identified an association between high miR-155-5p expression and a poor survival rate (n=73); miRNA-155-5p may be considered as a specific factor resulting in a worse prognosis (35).

Serum miR-483-5p was higher in patients with oral cancer (n=101) compared with healthy controls (n=103); the survival rates of patients with high miR-483-5p serum level (n=43; >3.23-fold higher compared with healthy controls) were decreased compared with that of patients with lower miR-483-5p serum levels (n=42; <3.23-fold higher compared with healthy controls), and multivariate analyses for overall survival suggested that a high miR-483-5p serum level was an independent prognostic indicator (36). Furthermore, patient follow-up revealed that patients with higher blood miR-372 levels had more extensive primary tumors, a greater tendency of node metastases, a more terminal stage and higher mortality rates (57). Additionally, miR-372 was downregulated in plasma and saliva among postoperative patients compared with preoperative patients (57). By measuring salivary miR-31, it was determined that 86.4% (19/22) of patients exhibited a significant decrease in miR-31 levels following tumor resection (28). miR-372 and miR-31 may therefore serve as biomarkers for the evaluation of surgical efficacy (28,57). Sun et al (58) suggested that serum miR-9 is an independent prognostic factor for oral cancer, as downregulated miR-9 was associated with lymph node metastases, advanced TNM stage and poor prognosis. Patients with low serum miR-9 expression had a worse disease-free survival rate compared with patients with high miR-9 expression (26.5 and 54.6%, respectively). The overall survival rate of patients with low and high miR-9 expression was 42.9 and 67.3%, respectively. By determining levels of miRNAs in the serum or saliva of patients, miRNAs may be used in minimally or non-invasive methods to predict lymph node metastasis and assess the prognosis of patients with oral cancer. Table I presents the aberrant levels of other miRNAs in the saliva, blood, serum and plasma (28–30,59–68).

As a salivary gland-derived malignancy, mucoepidermoid carcinoma may occur in the oral cavity. Shin et al (69) reported that the overexpression of miR-127-3p led to an increase in the number of cells in the G₁ phase, indicating that miR-127-3p resulted in G₁/S cell cycle arrest in vitro. miR-127-3p-induced cell cycle arrest in mucoepidermoid carcinoma MC-3 cells was associated with the increase of cyclin-dependent kinase inhibitor 1A and interferon α inducible protein 27 expression via the regulation of Sp1 transcription factor (69). Binmadi et al (70) found that miR-302a was significantly increased in mucoepidermoid carcinoma tissues compared with normal tissues. Furthermore, upregulated miR-302a induced invasion of mucoepidermoid carcinoma cells in vitro.

Adenoid cystic carcinoma (ACC) generally occurs in the minor salivary glands and has a poor long-term prognosis due to perineural invasion and lung metastasis (71). Wang et al (72) analyzed the expression of miR-130a in 21 patients with ACC and corresponding normal salivary gland tissues. Compared with that in normal salivary gland tissues, the expression of miR-130a in ACC tissues increased by 1.58–29.1 times. In addition, the level of miR-130a was negatively correlated with N-myc downstream-regulated gene 2 in ACC tissues. Chen et al (73) analyzed miRNAs during the metastasis of ACC cells and found that the expression levels of miR-4487, miR-4430 and miR-486-3p were upregulated, and the expression levels of miR-5191, miR-3131 and miR-211-3p were downregulated. Andreasen et al (74) found that high expression levels of miR-21, miR-181a-2 and miR-152 in patients with ACC was associated with a decreased overall survival rate, and high expression of miR-374c was associated with an improved relapse-free survival rate. Wang et al (75) found that an miR-21 inhibitor significantly reduced the resistance of lung metastatic salivary adenoid cystic carcinoma cells (SACC-LM) to simvastatin. Furthermore, the combination of simvastatin and miR-21 inhibitor decreased the proliferation of SACC-LM cells (75).

Yang et al (76) found that when a recombinant lentivirus carrying the miR-381-3p gene was transduced into SCC-9 and Tca-8113 cell lines, miR-381-3p-overexpressing cells demonstrated downregulation of fibroblast growth factor receptor 2. Furthermore, the percentage of cells at the G₁/G₀ phase was increased and the number of cells at the S phase was decreased. In addition, the apoptotic rate was significantly increased and the number of colonies was decreased in SCC-9 and Tca-8113 cells overexpressing miR-381-3p compared with those in the negative control group. EMT, an important mechanism of invasion or metastasis of cancer, which involves the downregulation of E-cadherin and increases metastasis and invasion as a consequence of loss of intercellular adhesion, may be regulated by miRNAs (77). ZEB was reported as an EMT-related transcription factor, which was shown to directly combine with the E-cadherin promoter and inhibit its transcription (77,78). Hashiguchi et al (79) identified an association between miR-205 and the EMT phenotype in SQUU-B cells and demonstrated that overexpression of miR-205 downregulated ZEB1, ZEB2 and N-cadherin, and upregulated E-cadherin. Another study reported that overexpression of miR-375 significantly upregulated SCC-4 cell radiation-induced apoptosis by directly regulating the insulin-like growth factor 1 receptor (IGF1R) (32). Tumor suppressor miRNAs, and their respective target genes and downstream signaling pathways, are presented in Table II (80–111).

Fu et al (49) demonstrated that miR-155 targeted the cyclin-dependent kinase inhibitor 1B (CDKN1B) 3′-UTR by a luciferase reporter assay. Furthermore, downregulation of miR-155 led to an increase in CDKN1B and inhibited cell proliferation and cell cycle progression in oral cancer Tca8113 cells (49). A previous study demonstrated that the mRNA and protein levels of vimentin and N-cadherin were upregulated in SCC-9 and UM1 cells transfected with miR-373-3p, respectively, while E-cadherin and CK18 were downregulated, suggesting that miR-373-3p may stimulate the EMT phenotype (48). The repression of the negative regulator of the Wnt signaling pathway, Dickkopf-related protein 1, and the nuclear accumulation of β-catenin promoted the Wnt signaling pathway, which facilitated proliferation of SCC-9 and UM1 cells (48). Dysregulation of the Wnt signaling pathway in tumors may result in chemoresistance. Protein phosphatase 2 regulatory subunit Bα (PPP2R5A) is a repressor of Wnt signaling, and in oral cancer cells, miR-218 increased cisplatin resistance via the Wnt signaling pathway by repressing PPP2R5A expression (112). Zhuang et al (112) demonstrated that when UM1 and Cal27 cells were transfected with miR-218 mimics, the miR-218 overexpression resulted in increased levels of β-catenin, indicating activation of Wnt signaling, and enhanced cell viability. Furthermore, blocking the effect of miR-218 reversed chemoresistance in resistant cells. Jiang et al (113) revealed that low expression of miR-222 increased the chemosensitivity of oral cancer cells to cis-diaminedichloroplatinum (CDDP) and that the combination of antisense-miR-222 and CDDP may be an effective curative strategy by upregulating the expression of p53, a modulator of apoptosis. Table III presents tumor-promoting miRNAs with their respective target genes and downstream signaling pathways (112–129).

Bioinformatics analysis may be used to predict the pairing sequences of miRNAs and target genes, which may be verified by luciferase reporter assays. Western blotting and RT-qPCR may subsequently be used to detect protein and miRNA expression, respectively. Frequently used oral cancer cell lines include SCC-9, SCC-4, Tca-8113 and Cal27 (45,48,49,51). Cell experiments demonstrated that specific miRNAs served an anticancer role such as miR-138, miR-200c, miR-15b, miR-485-5p and miR-340 (83,87,89,91,109); whereas other miRNAs stimulated oral cancer by regulating cellular proliferation, apoptosis, invasion, EMT, metastasis, radiosensitivity, chemosensitivity and glucose metabolism, such as miR-221, miR-455-5p, miR-27a-3p, miR-21 and miR-10a (114,117,126,128,130). The results obtained from in vitro cell experiments may provide novel therapeutic targets for potential clinical application.

miRNAs are promising therapeutic targets, as they serve important roles in cancer. Theoretically, by silencing tumor-promoting miRNAs and inducing the expression of tumor-suppressing miRNAs synchronously, it is possible to treat oral carcinoma. Specific miRNAs have multiple target genes, for example, miR-375 targets IGF1R (32), platelet-derived growth factor subunit A (131), Kruppel-like factor 5 (80) and solute carrier family 7 member 11 (81); miR-138 targets AKT serine/threonine kinase 1 (82) and yes-associated protein 1 (83); miR-203 targets B lymphoma Mo-MLV insertion region 1 homolog (84), semaphorin 6A (85) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (86); and miR-221 targets tissue inhibitor of metalloproteinase 3 (114) and phosphatase and tensin homolog (PTEN) (115). A possible treatment strategy may involve anticancer drugs that selectively regulate the expression of such miRNAs. Certain genes or signaling pathways are regulated by two or more miRNAs, for instance, tripartite motif containing 14 is targeted by miR-195-5p (31) and miR-15b (89); IGF1R is targeted by miR-98 (132) and miR-375 (32); ZEB1 is targeted by miR-205 (79) and miR-200c (87); and PTEN is targeted by miR-221/222 (115) and miR-24 (116). Therefore, the combination of more specific inhibitors or activators of cancer-associated genes or signaling pathways may be a suitable therapeutic strategy. With regard to controversial miRNAs, including miR-222, which served an ambivalent role in disparate experiments, more research is required to verify their roles in oral cancer (88,113,115).