The OSCC tissues and adjacent normal oral epithelial tissues were collected from 40 patients with OSCC at the Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Xinxiang Medical University (Weihui, China) between November 2015 and January 2016. The patients enrolled in this study did not receive radiotherapy or chemotherapy prior to surgical resection. All tissue samples were confirmed by pathological examination. All OSCC tissues and corresponding normal oral epithelial tissues were frozen in liquid nitrogen and stored at −80°C until further use. Patient characteristics are presented in Table I. The present study was approved by the Ethics Committee of The First Affiliated Hospital of Xinxiang Medical University and all patients provided written informed consent.

The human OSCC cell lines HSC-2, CAL-27, Tca8113 and SCC-4 were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were grown in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 100 IU/ml penicillin and 100 mg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. The normal human oral keratinocytes (HOK) (ScienCell Research Laboratories, Inc., San Diego, CA, USA) were grown in oral keratinocyte medium (Invitrogen; Thermo Fisher Scientific, Inc.).

miR-199a-5p mimics/inhibitor and the corresponding negative control (NC) sequences (mimics/inhibitor NC) were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). Small interfering (si)RNA sequences, si-SOX4 and NC si-Scramble, were purchased from Ambion (Thermo Fisher Scientific, Inc.). The oligonucleotides sequences as the following: miR-199a-5p mimics, sense 5′-CCCAGUGUUCAGACUACCUGUUC-3′; mimics NC, sense 5′-CGGTGUGUUCAGACUACCUGUUC-3′; miR-199a-5p inhibitor, sense 5′-AACAGGTAGTCTGAACACT-3′; inhibitor NC, sense: 5′-TAACACGTCTATACGCCCA-3′; si-SOX4-1, 5′-GCAAACGCTGGAAGCTGCTCAAAGA-3′; si-SOX4-2, 5′-TGGAAGCTGCTCAAAGACAGCGACA-3′; si-SOX4-3, 5′-GGAAGCTGCTCAAAGACAGCGACAA-3′; and si-Scramble,: 5′-TGGGTCGACTCAGAACGACGAAACA-3′. For plasmid construction (pcDNA-SOX4), the SOX4 3′-untranslated region (UTR) was cloned into the XhoI and KpnI sites of the pcDNA3.1 expression vector (Invitrogen; Thermo Fisher Scientific, Inc.); an empty pcDNA3.1 vector (pcDNA-vector) used as a control. For transfection, SCC-4 or HSC-2 cells were plated in 6-well plates (2×10⁶ cells/well) and were transfected with miR-199a-5p mimics/inhibitor, mimics/inhibitor NC, si-SOX4/si-Scramble or pcDNA-SOX4/pcDNA-vector at a final concentration of 100 nM using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Subsequently, cells were cultured at 37°C in an incubator containing 5% CO₂ for 48 h after trans-fection, after which, cells underwent subsequent experiments. Transfection efficiency was assessed using RT-qPCR.

Total RNA was isolated from tissues and cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. TaqMan Gene Expression Assay and TaqMan MicroRNA Reverse Transcription kits (Applied Biosystems; Thermo Fisher Scientific, Inc.) were used for RT prior to SOX-4 and miRNA detection, according to the manufacturer's protocol. PCR was performed on an ABI PRISM 7300 sequence detection system using a SYBR-Green I Real-Time PCR kit (both Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR amplification was conducted at 95°C for 3 min, followed by 35 cycles comprising 95°C for 10 sec, 60°C for 30 sec and 72°C for 30 sec. U6 served as an endogenous control for miR-199a-5p and GAPDH served as a reference control for SOX4. The following primers were used: miR-199a-5p, forward 5′-TCCCAGTGTTCAGACTACC-3′, reverse 5′-TTTGGCACTAGCACATT-3′; SOX4, forward 5′-CCAGTTCTTGCACGCTGTTT-3′, reverse 5′-TGTTGCAAGGTAGGAAGCCA-3′; U6, forward 5′-CTCGCTTCGGCAGCACA-3′, reverse 5′-AACGCTTCACGAATTTGCGT-3′; and GAPDH, forward 5′-AACGTGTCAGTGGTGGACCTG-3′ and reverse 5′-AGTGGGTGTCGCTGTTGAAGT-3′. Fold changes were determined using the relative quantification (2^−ΔΔCq) method (26).

Cell invasion assays were performed using 24-well Transwell chambers with polycarbonate membranes (pore size, 8.0 µm; Corning Inc., Corning, NY, USA). Briefly, SCC-4 or HSC-2 cells (2×10⁶ cells/well) were plated into the upper chambers, which were precoated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). After 48 h at 37°C, non-invading cells on the upper side of the membrane were removed and invasive cells on the lower surface of the membrane were fixed with 20% methanol at 4°C for 30 min and stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA). The number of invasive cells was counted from six random fields in each well using an IX71 inverted microscope (Olympus Corporation, Tokyo, Japan); images were then captured (magnification, ×200).

A wound-healing assay was performed to analyze cell migration. Briefly, SCC-4 or HSC-2 cells (2×10⁶ cells/well) were plated into 6-well plates. After 48 h at 37°C, the migration status was examined by measuring the movement of cells into a scraped area, which was created using a micropipette tip. After 0 or 48 h, cells were fixed in 4% paraformaldehyde (cat. no. 15714; Electron Microscopy Sciences) for 1 h at room temperature, and were observed under a microscope. The wounds were analyzed in six random fields using an IX71 inverted microscope (Olympus Corporation; magnification, ×200). The percentage of closure of the denuded area was calculated using ImageJ software (version 1.44; National Institutes of Health, Bethesda, MD, USA).

Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8) assay, according to the manufacturer's protocol. Briefly, SCC-4 or HSC-2 cells were seeded into 96-well plates at an initial density of 5×10⁴ cells/well, and were cultured in 100 µl RPMI-1640 medium supplemented with 10% FBS. At the specified time points (24, 48 and 72 h), 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) was added to each well. Subsequently, the plate was incubated for 1 h at 37°C in an atmosphere containing 5% CO₂ and the absorbance rate was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Experiments were performed in triplicate.

SCC-4 or HSC-2 cells were lysed as described previously (2) and the protein concentrations were measured using a bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Protein samples (30 µg) were then separated by 10% SDS-PAGE (Sigma-Aldrich; Merck KGaA) and were transferred onto polyvinylidene difluoride (PVDF) membranes (GE Healthcare Life Sciences, Little Chalfont, UK). The PVDF membranes were blocked in 5% nonfat milk (Sigma-Aldrich; Merck KGaA) at room temperature for 1 h and were then incubated with the following primary antibodies at 4°C overnight: SOX-4 (1:500; cat. no. ab80261; Abcam, Cambridge, MA USA), E-cadherin (1:1,000; cat. no. sc-7870; Santa Cruz Biotechnology, Inc., Dallas, TX. USA), N-cadherin (1:1,000; cat. no. ab18203; Abcam), fibronectin (1:1,000; cat. no. sc-18825; Santa Cruz Biotechnology, Inc.) and vimentin (1:1,000; cat. no. sc-5565; Santa Cruz Biotechnology, Inc.). β-actin (1:750; cat. no. A5060; Sigma-Aldrich; Merck KGaA) served as an internal control. Membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG secondary antibodies [dilution 1:10,000; cat. nos. GAR0072 and GAM0072; MultiSciences (Lianke) Biotech, Co., Ltd., Hangzhou, China] at room temperature for 2 h. Subsequently, the protein bands were scanned on X-ray film and were visualized using the enhanced chemiluminescence detection system (PerkinElmer, Inc., Waltham, MA, USA). The relative intensity of each band was semi-quantified using Alpha Imager software version 2000 (ProteinSimple, San Jose, CA, USA). The measurements were conducted independently at least three times with similar results.

Bioinformatics analysis (TargetScan; www.targetscan.org/vert_72) was used to predict the putative target genes of miR-199a-5p in OSCC cells. The SOX4 3′-UTR encompassing the target sequence for miR-199a-5p was amplified by PCR and cloned into the psi-CHECK-2 Vector (Promega Corporation, Madison, WI, USA) (wild type psi-CHECK-2-SOX4-3′-UTR). Site-directed mutagenesis of the miR-199a-5p target site in the SOX4 3′-UTR (psi-CHECK-2-SOX4-mut-3′-UTR) was performed using the QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA). SCC-4 cells (5×10⁴ cells/well) were seeded into 96-well plates and the cells were co-transfected with 400 ng psi-CHECK-2-SOX4-3′-UTR or psi-CHECK-2-SOX4-mut-3′-UTR, and 50 ng miR-199a-5p mimics/inhibitor or the corresponding NC using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The ratio of firefly to Renilla luciferase was used to normalize relative firefly luciferase activity 48 h post-transfection, and luciferase activity was measured using the Dual-Luciferase^® Reporter Assay system (Promega Corporation), according to the manufacturer's protocol.

All statistical analyses were carried out using SPSS software (version 18.0; SPSS, Inc., Chicago, IL, USA). Data are presented as the means ± standard deviation. Comparisons between two groups were made using Student's t-test. A one-way analysis of variance followed by Tukey's post-hoc multiple comparisons test was used to determine statistically differences among multiple groups. The associations between miR-199a-5p level and clinicopathological features were analyzed using χ² test. Survival analysis was conducted using the Kaplan-Meier method followed by a log-rank (Mantel-Cox) test. The correlation between miR-199a-5p and SOX4 expression was assessed by Spearman's analyses. P<0.05 was considered to indicate a statistically significant difference.

To investigate the association between miR-199a-5p expression and OSCC, RT-qPCR was used to detect miR-199a-5p expression in 40 paired OSCC tissues and adjacent normal oral epithelial tissues. As shown in Fig. 1A, miR-199a-5p expression was markedly downregulated in OSCC tissues compared with in normal oral epithelial tissues (P<0.01). Furthermore, miR-199a-5p expression was reduced in OSCC tissues with lymph node metastasis (n=28) compared with in tissues without lymph node metastasis (n=12) (P<0.01; Fig. 1B). This study further analyzed the association between miR-199a-5p and the clinicopathological features of patients with OSCC (Table I). To determine the clinical value of miR-199a-5p, the mean expression level of miR-199a-5p was used as a cut-off value to divide 40 patients with OSCC into two groups: The miR-199a-5p high expression group and the miR-199a-5p low expression group. The results revealed that low miR-199a-5p expression in OSCC was associated with lymph node metastasis (P=0.003), differentiation (P=0.037) and tumor size (cm) (P=0.020) (Table I). However, miR-199a-5p expression was not associated with sex, age (years), tumor site, alcohol use, smoking and clinical Tumor-Node-Metastasis stage (27) (Table I). Kaplan-Meier analysis indicated that OSCC patients with low miR-199a-5p expression (n=24) had a poorer overall survival rate compared with patients with high miR-199a-5p expression (n=16) (P<0.01; Fig. 1C). In addition, this study further confirmed that miR-199a-5p was markedly downregulated in the OSCC cell lines HSC-2, CAL-27, Tca8113 and SCC-4 compared with in HOK cells (P<0.01; Fig. 1D). The two OSCC cell lines, HSC-2 and SCC-4, with the lowest expression levels of miR-199a-5p, were selected for subsequent analysis in the present study. These data indicated that miR-199a-5p may serve an important role in the development of OSCC.

To further elucidate the role of miR-199a-5p in OSCC cells, SCC-4 and HSC-2 cells were transfected with miR-199a-5p mimics or mimics NC, and cell invasion and migration were evaluated using Transwell invasion and wound-healing assays, respectively. This study initially evaluated the transfection efficiency of miR-199a-5p mimics and miR-199a-5p inhibitor in SCC-4 and HSC-2 cells. The results revealed that miR-199a-5p expression was markedly upregulated in SCC-4 and HSC-2 cells transfected with miR-199a-5p mimics compared with in cell transfected with mimics NC, whereas transfection with miR-199a-5p inhibitor downregulated miR-199a-5p expression compared with inhibitor NC (P<0.01; Fig. 2A and B). Subsequently, the results revealed that miR-199a-5p overexpression significantly inhibited the invasion of SCC-4 and HSC-2 cells compared with mimics NC (P<0.01; Fig. 2C and D). Furthermore, the results further confirmed that overexpression of miR-199a-5p markedly suppressed cell migration compared with mimics NC (P<0.01; Fig. 2E and F). These data suggested that miR-199a-5p may exert suppressive effects on cell migration and invasion in OSCC.

Tumor cell invasion and metastasis are tightly correlated with various processes, including EMT. During EMT, epithelial tumors progress to more aggressive metastatic tumors, alongside which the expression of mesenchymal protein markers, including N-cadherin and vimentin, is upregulated, whereas the epithelial protein marker E-cadherin is downregulated (14,28-29). It has been well documented that miRNAs regulate tumorigenesis via modulating EMT in various types of cancer, including OSCC (30-32). The present results demonstrated that miR-199a-5p upregulated the expression levels of E-cadherin, and downregulated N-cadherin, vimentin and fibronectin in SCC-4 and HSC-2 cells (Fig. 2G). Taken together, these results suggested that miR-199a-5p may inhibit cell invasion and migration in OSCC cells via modulating EMT.

Bioinformatics analysis revealed that SOX4 may be a potential target gene of miR-199a-5p in OSCC cells (Fig. 3A). Previous studies have reported that SOX4 functions as an oncogene in numerous types of cancer, such as prostate cancer, lung cancer and hepatocellular carcinoma (9-11). Additionally, SOX4 has been identified to serve a key role in EMT and may induce cancer cell metastasis (1,13). Therefore, to further investigate the association between miR-199a-5p and SOX4 in OSCC, SOX4 expression was detected in OSCC tissues and adjacent normal oral epithelial tissues. As shown in Fig. 3B, SOX4 expression was significantly upregulated in tumor tissues compared with in normal tissues (P<0.01). Correlation analysis revealed that there was a significant negative correlation between miR-199a-5p and SOX4 expression in 40 tumor tissues (r=-0.7441, P<0.01; Fig. 3C). Furthermore, overexpression of miR-199a-5p markedly inhibited SOX4 (P<0.01; Fig. 3D), whereas inhibition of miR-199a-5p significantly upregulated SOX4 expression in SCC-4 and HSC-2 cells (P<0.01; Fig. 3E). Luciferase reporter assay revealed that miR-199a-5p markedly inhibited luciferase activity when compared with mimics NC, whereas miR-199a-5p inhibitor significantly enhanced luciferase activity (P<0.01; Fig. 3F). However, miR-199a-5p mimic/inhibitor did not affect luciferase activity in cells transfected with psi-CHECK-2-SOX4-mut-3′-UTR (Fig. 3F). Collectively, these results indicated that miR-199a-5p may suppress SOX4 expression by targeting its 3′-UTR in OSCC.

To explore the role of SOX4 in OSCC cells, SCC-4 and HSC-2 cells were transfected with si-SOX4 or si-Scramble, and cell proliferation, invasion and migration were evaluated using CCK-8, Transwell invasion and wound-healing assays, respectively. Firstly, the effects of si-SOX4 (si-SOX4-1, si-SOX4-2 and si-SOX4-3) on SOX4 expression in OSCC cells were analyzed. As shown in Fig. 4A and B, all three siRNAs significantly inhibited SOX4 expression in SCC-4 and HSC-2 cells compared with si-Scramble (P<0.01); si-SOX4-1 exhibited the strongest effect and was selected for subsequent experiments. As shown in Fig. 4C and D, knockdown of SOX4 markedly suppressed proliferation of SCC-4 and HSC-2 cells compared with si-Scramble (P<0.05). Furthermore, cell invasion and migration were significantly inhibited in SCC-4 and HSC-2 cells transfected with si-SOX4 (P<0.01; Fig. 4E-G). These results indicated that SOX4 may act as an oncogene in OSCC via modulating cell proliferation, migration and invasion.

To further investigate whether SOX4 is a functional target of miR-199a-5p in OSCC cells, SCC-4 and HSC-2 cells were transfected with miR-199a-5p mimics/mimics NC or were co-transfected with miR-199a-5p mimics and pcDNA-SOX4/pcDNA-vector plasmids, and cell invasion and migration were determined using Transwell invasion and wound-healing assays, respectively. Firstly, the transfection efficiency of pcDNA-SOX4 was evaluated in SCC-4 and HSC-2 cells; SOX4 was significantly upregulated compared with in cells transfected with the pcDNA-vector (P<0.01; Fig. 5A). Subsequently, the results demonstrated that overexpression of miR-199a-5p markedly suppressed cell migration; however, upregulation of SOX4 significantly increased miR-199a-5p-mediated migration of SCC-4 and HSC-2 cells (P<0.01; Fig. 5B and C). Furthermore, the results confirmed that the suppressive effect of miR-199a-5p on cell invasion was rescued by SOX4 in SCC-4 and HSC-2 cells (P<0.01; Fig. 5D and E). These data suggested that the suppressive effects of miR-199a-5p on cell migration and invasion may be rescued by SOX4 overexpression in OSCC cells.