The CAL-27, Tca8113, UM1 and UM2 OSCC cell lines (China Center for Type Culture Collection, Wuhan, China) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). All cells were incubated at 37°C in 5% CO₂.

miR-negative control (miR-NC, 5′-UCACAACCUCCUAGAAAGAGUAGA-3′) and miR-375 mimic (5′-UUUGUUCGUUCGGCUCGCGUGA-3′) were purchased from GenePharma Co., Ltd. (Shanghai, China). The full-length PDGF-A (NM_002607.5) was cloned and inserted into the pcDNA3.1 expression plasmid (Promega Corporation, Madison, WI, USA). Cells were plated at 50% confluence and transfected with 300 nM miR-NC, miR-375 mimic and/or 10 µM PDGF-A or pcDNA3.1 empty vector using the Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Cells were harvested 24 or 48 h after transfection and subjected to further analysis.

Total RNA was extracted from cultured cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. miR-375 expression levels were analyzed using the TaqMan Advanced miRNA assay (Applied Biosystems; Thermo Fisher Scientific, Inc.) and U6 served as an internal control. To evaluate PDGF-A mRNA expression levels, RT-qPCR was performed using the PrimeScript RT Reagent kit and SYBR^® Premix Ex Taq reagent (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. GAPDH served as an internal control. RT-qPCR was performed on an Applied Biosystems 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PDGF-A and GAPDH primer sequences used for RT-qPCR are presented in Table I. Gene expression levels were measured in triplicate, quantified using the 2-^ΔΔCq method, and normalized to the control (13). The qPCR was conducted using the following conditions: 95°C for 5 min followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec.

Cell migration and invasion were assessed using a Transwell migration assay. For this, UM1 cells were harvested, and 5×104 cells in 200 µl DMEM containing 0.1% FBS were placed into the upper chamber of an insert (8-µm pore size; BD Biosciences, San Jose, CA, USA). The lower chamber was filled with 600 µl DMEM containing 10% FBS. Migrating cells were imaged using a Leica light microscope (Leica Microsystems GmbH, Wetzlar, Germany; magnification, ×200) in five randomly selected fields per well, and the mean count was calculated. For the invasion assays, 5×104 cells were seeded into the upper chamber, which had been precoated with Matrigel (BD Biosciences) and incubated for 24 h. Following the removal of cells from the upper chamber with a cotton swab, the cells in the lower chamber were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet solution in 20% ethanol, and counted in five randomly-selected fields, using a phase contrast light microscope at a magnification ×200 (Olympus Corporation, Tokyo, Japan), following which the mean count was calculated. The assays were performed in triplicate.

The different experimental groups of UM1 cells were harvested and lysed using radioimmunoprecipitation assay buffer (Takara Biotechnology Co., Ltd.) and the total protein concentration was determined using the Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology, Nantong, China). Proteins (30 µg) were separated by 8% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. After blocked with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h at 37°C, the membranes were incubated with a polyclonal rabbit anti-human PDGF-A antibody (1:500; cat. no. ab125268; Abcam, Cambridge, MA, USA) or a monoclonal rabbit anti-human β-actin antibody (1:2,000; cat. no. ab115777; Abcam) for 1 h at 37°C. Membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG H&L secondary antibody (1:10,000; cat. no. ab97080; Abcam) for 40 min and proteins were visualized using an Enhanced Chemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc.). The expression levels of the proteins of interest were normalized against the expression levels of β-actin. Quantification was performed using ImageJ version 6.0 (National Institutes of Health, Bethesda, MD, USA).

miR-375 targets were predicted using the online miRNA target prediction software TargetScan (www.targetscan.org) and miRanda (www.microrna.org/microrna/home.do). To determine whether the 3′-UTR of PDGF-A mRNA was a direct target of miR-375, the full-length wild-type 3′-UTR of PDGF-A and mutant 3′-UTR of PDGF-A were amplified and cloned into the psi-CHECK-2 vector (Promega Corporation, Madison, WI, USA). The primer sequences used for the construction of the reporter vector are presented in Table I. UM2 cells were co-transfected with 100 ng plasmid and 200 nmol/l miR-375 mimic or miR-NC in 24-well plates. Cell lysates were harvested 48 h after transfection. The firefly and Renilla luciferase fluorescence intensities were measured using the Dual-Luciferase Reporter assay system (Promega Corporation) and experiments were performed in triplicate.

All statistical analyses were performed using SPSS software version 19.0 (IBM SPSS, Armonk, NY, USA). Data are presented as the mean ± standard deviation. A one-way analysis of variance was used to compare the differences between miR-375 expression levels in different OSCC lines and followed by a least significant difference post hoc test. A Student's t-test was used to compare the differences prior to and following treatment. P<0.05 was considered to indicate a statistically significant difference.

To assess the role of miR-375 in the regulation of OSCC cell migration and invasion, miR-375 expression levels in UM2 and Tca8113 (less metastatic) and UM1 and CAL-27 (highly metastatic) OSCC cell lines were determined using RT-qPCR. The results demonstrated that expression levels of miR-375 were significantly decreased in UM1 compared with Tca8113 (P<0.001) and UM2 cell lines (P=0.003); however, no significant differences were identified in the CAL-27 cell line (P=0.278; Fig. 1). The expression levels of miR-375 were reduced in the highly metastatic OSCC cell lines, particularly in UM1, compared with the less metastatic ones. Based on these results, the UM1 cell line was selected for further experiments.

To assess the role of miR-375 in the regulation of OSCC cell migration and invasion, an miR-375 mimic or an miR-NC was transfected into UM1 cells. After 48 h of culture, UM1 cells were harvested for RT-qPCR. The results demonstrated that transfection of UM1 cells with an miR-375 mimic significantly increased miR-375 expression levels in UM1 cells (P=0.005) (Fig. 2A). Furthermore, following the transfection of cells with the miR-375 mimic or miR-NC for 48 h, Transwell assays were performed to measure UM1 cell migration and invasion. The Transwell migration assay revealed that the ability of the cells to migrate through the membrane into the lower chamber was significantly inhibited in the miR-375 mimic-transfected cells compared with miR-NC-transfected cells (P=0.002; Fig. 2B). The Transwell invasion assay demonstrated that the ability of the cells to pass through a Matrigel-coated membrane into the lower chamber was significantly decreased in the miR-375 mimic-transfected cells compared with the miR-NC-transfected cells (P<0.001; Fig. 2C). Representative images of migrated and invaded cells are presented in Fig. 2D and E, respectively.

To further elucidate the mechanisms underlying miR-375-mediated suppression of UM1 cell migration and invasion, the targets of miR-375 were identified using the prediction algorithms of TargetScan and miRanda. This analysis identified PDGF-A as a potential target of miR-375, based on the putative binding site at positions 163–169 of the PDGF-A 3′-UTR (Fig. 3A). To demonstrate the association between miR-375 and PDGF-A, the protein expression levels of PDGF-A in the UM1, CAL-27, UM2 and Tca8113 cell lines were determined. The results confirmed that the UM1 and CAL-27 (highly metastatic) cell lines had greater levels of PDGF-A protein compared with the Tca8113 and UM2 (less metastatic) cell lines (P<0.001, Tca8113 and UM2 compared with UM1; P=0.003, P=0.009, respectively; Tca8113 and UM2 compared with CAL-27; Fig. 3B and C). Luciferase reporter vectors, co-transfected into UM1 cells with miR-375 mimic or miR-NC, were used to further examine whether PDGF-A was the target of miR-375. These contained wild-type PDGF-A 3′-UTR or a mutant 3′UTR mutated at the predicted miR-375 binding site. A significant decrease in the luciferase activity of the reporter was observed for the wild-type PDGF-A 3′-UTR-containing vector compared with miR-NC (P<0.001), whereas this decrease did not occur when the target site was the mutated PDGF-A 3′-UTR (P=0.158; Fig. 3D). These results indicated that the sequence in the 163–169 bp region of the PDGF-A 3′-UTR interacted with miR-375, leading to the inhibition of PDGF-A expression levels in UM1 cells. Additionally, the effects of miR-375 overexpression on PDGF-A mRNA and protein levels were examined. As expected, overexpression of miR-375 did not cause degradation of PDGF-A mRNA (P=0.096) (Fig. 3E); however, it did result in a significant reduction in the protein expression levels of PDGF-A (P=0.008; Fig. 3F and G). These data indicated that miR-375 directly targeted the 3′-UTR of PDGF-A.

To determine whether miR-375 contributes to UM1 migration and invasion via PDGF-A, UM1 cells were transfected with a PDGF-A expression vector for 48 h. PDGF-A protein expression levels were increased in cells co-transfected with the PDGF-A expression vector and miR-375 mimic, compared with cells co-transfected with the NC vector and miR-375 mimic (P<0.001; Fig. 4A and B). Transwell migration and invasion assays were performed, and representative images are presented in Fig. 4C and D, respectively. The Transwell migration assay demonstrated that the ability of these cells to migrate through the membrane into the lower chamber was significantly enhanced in miR-375 mimic- and PDGF-A-transfected cells compared with miR-375 mimic- and NC-transfected cells (P=0.001; Fig. 4E). Additionally, the Transwell invasion assays revealed that the ability of the cells to pass through the Matrigel-coated membrane into the lower chamber was significantly increased in miR-375 mimic- and PDGF-A-transfected cells compared with miR-375 mimic- and NC-transfected cells (P<0.001; Fig. 4F). These results suggested that miR-375 affected the migration and invasion of UM1 cells via regulation of its target gene, PDGF-A, and that overexpression of PDGF-A was able to reverse the suppressive effect of miR-375 in UM1 cells.