The SACC83 SACC cell line [kindly provided by Professor Shenglin Li (Department of Oral and Maxillofacial Surgery, School of Stomatology, Beijing University, Beijing, China)] was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; GE Healthcare Life Sciences, Logan, UT, USA), 50,000 units penicillin and 50 mg streptomycin (both from Sigma-Aldrich, St. Louis, MO, USA) at 37°C in a humidified incubator containing 5% CO₂.

To determine the dose- and time-dependent changes associated with EGCG treatment, the SACC83 cells were treated in triplicate with EGCG (Wako Pure Chemical Industries, Ltd., Osaka, Japan), as described previously (19). Briefly, the cells were seeded at low density (5×10⁵/100 mm dish) 24 h prior to treatment with EGCG. To determine the dose-dependent changes, the cells were treated with 0, 5, 15 or 45 µm EGCG for 6 days. EGCG was added, in new culture medium, to the cells on days 1, 3 and 5. For the assessment of time-dependent changes, the cells were treated with 45 µm EGCG for 0, 36, 72 or 144 h.

Genomic DNA was isolated and modified using a CpGenome™ Direct Prep Bisulfite Modification kit (EMD Millipore, Billerica, MA, USA), according to the manufacturers' instructions. The DNA concentration was determined using spectrophotometry. For MSP, the following primer sets (Takara Bio Inc., Dalian, China) were used for methylated DNA: M_RECK, forward 5′-ATAAAGAGTTTTGGTACGGGGTAC-3′ and reverse 5′-AAAACCGCGAAATACTCGAA-3′); and the primer sets for unmethylated DNA were as follows: U_RECK, forward 5′-TAAAGAGTTTTGGTATGGGGTATGT-3′ and reverse 5′-CTCCAAACCACAAAATACTCAAA-3′. The MSP reactions were performed in a mixture of 12.5 µl 2X Taq PCR Master mix (Tiangen Biotech, Co., Ltd., Beijing, China) reverse primers (1 µl), DNA (<1 µg) and distilled water in 25-µl volumes in a S1000 (Bio-Rad Laboratories, Inc., Hercules, CA. USA) under the following conditions: 94°C for 3 min; followed by 30 cycles at 94°C for 30 sec, 55°C for 30 sec and 72°C for 60 sec; and finally 5 min at 72°C. The PCR product lengths for the methylated and unmethylated RECK were 195 and 199 bp, respectively. Universal Methylated DNA (EMD Millipore) and normal human blood DNA were used as positive controls for methylated and unmethylated statuses, respectively. The blood was donated by the authors of the present study, and ethical approval was obtained from the ethics committee of Weifang People's Hospital (Weifang, China). A water blank was used as a negative control. The positive and negative controls were used appropriately in each round of MSP. All assays were performed in triplicate. The PCR products were separated on 2% agarose gels (Zeta Biotechnology, Co., Ltd., Guangzhou, China) and visualised using ethidium bromide (EtBr; Haoran Biological Technology Co., Ltd., Shanghai, China) staining.

Total RNA was extracted using a Total RNA Extraction kit (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's instructions. The quantity and quality of the RNA samples were measured using a spectrophotometer and electrophoresis. RECK cDNA was synthesised from 1 µg of total RNA using a PrimeScript™ RT Reagent kit with gDNA Eraser (Takara Biotechnology Co., Ltd.). The following primers (Takara Bio Inc.) were used for PCR: RECK, forward 5′-CCTCAGTGAGCACAGTTCAGA-3′ and reverse 5′-GCAGCACACACACTGCTGTA-3′. Reactions were performed in 20 µl volumes containing 2X Mater Mix (10µl), forward and reverse primers (1 µl), cDNA (2 µl) and distilled water under the following conditions: 95°C for 10 min; followed by 35 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec; and finally 7 min at 72°C. GAPDH was used as an internal control to estimate the efficiency of cDNA synthesis using the primer: GAPDH, forward 5′-GCAGCACACACACTGCTGTA-3′ and reverse 5′-TGTGGTCATGAGTCCTTCCA-3′. The predicted size for the PCR products of RECK and GAPDH were 477 and 512 bp, respectively. The PCR products were separated on 2% agarose gel, stained with EtBr and visualised under ultraviolet light and images were captured using a Bio-Rad VersaDoc 3000 Imaging system (Bio-Rad Laboratories, Inc.). Image J software (version 1.48u; National Institutes of Health, Bethesda, MD, USA) was used for grey value analysis. The relative mRNA expression levels of RECK were normalised against GAPDH.

Following treatment with EGCG, the SACC83 cells were washed twice with cold phosphate-buffered saline (PBS) and treated with extraction buffer [50 M Tris-HCl (pH 7.4), 150 M NaCl, 2 M EDTA and 1% NP-40]. The cell extractions were subsequently centrifuged at 12,000 × g for 15 min at 4°C, and the supernatants were collected. The protein concentration was quantified using a Bicinchoninic Acid Protein Measurement kit (Shenneng Bocai Biology, Co., Ltd., Shanghai, China). Equal quantities (40 µg) of cellular proteins were subjected to 8% SDS-PAGE, and transferred onto polyvinylidene fluoride membranes (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The membranes were blocked with 5% (w/v) non-fat milk in PBS containing 0.1% Tween-20 (Shenneng Bocai Biology, Co., Ltd., Shanghai, China) and then incubated with primary antibodies (mouse anti-human RECK polyclonal, cat. no. ab88249; anti-GAPDH, cat. no. sc-365062) at 1:1,000 and room temperature for 1 h. Subsequently, the membranes were incubated with an appropriate horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G secondary antibody (cat. no. sc-2005) at room temperature for 1 h. The immuno-detected proteins were visualised using enhanced chemiluminescence. Image J software was used for grey value analysis. The anti-RECK antibody was purchased from Abcam (Cambridge, MA, USA) and the anti-GAPDH and secondary antibodies were obtained from Santa Cruz Biotechnology, Inc.

To assess the invasive ability of the cells treated with EGCG, Matrigel invasion assays were performed using 8-mm pore filter inserts in 24-well plates (Sigma-Aldrich) coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). The cells were incubated with EGCG (0, 5, 15 or 45 µm) for 72 h, and were subsequently collected, washed three times with PBS and re-suspended in serum-free DMEM. A total of 1×10⁵ cells/200 µl medium were plated in the upper chamber of the Transwell unit and allowed to invade for 24 h at 37°C. The lower chamber of the Transwell unit was filled with 500 µl medium supplemented with 10% FBS. At the end of the incubation period, the non-invaded cells on the upper surface of the membrane were carefully removed using a cotton swab. The invaded cells on the bottom surface of the membrane were fixed in 4% formaldehyde for 20 min and stained with 0.1% crystal violet for 5 min. Subsequently, the invaded cells on the lower surface of the membrane were visualised in five randomly-selected fields under a microscope (Olympus BH2; Olympus, Tokyo, Japan; magnification, ×200). All assays were performed in triplicate and the mean number of invaded cells was used for analysis.

Statistical analysis was performed using SPSS 13.0 for Windows (SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation. Differences between the groups were assessed using one-way analysis of variance with Dunnett's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

The RECK gene in SACC83 cells exhibited marked expression of methylated and weak expression of unmethylated promoter, determined using MSP (Fig. 1A). Normal human blood DNA was used as a positive control for unmethylation, and Universal Methylated DNA was used as a positive control for methylation. H₂O was used as a negative control.

The present study aimed to examine the time-and dose-dependent effects of EGCG in SACC83 cells. Following treatment of the cells with 0, 5, 15 or 45 µm EGCG for 144 h, methylation-specific bands of the RECK gene were weak in appearance, whereas unmethylation-specific bands of the RECK gene appeared markedly enhanced. Following treatment of the cells with 45 µm EGCG for 0, 36, 72 or 144 h, the unmethylation-specific bands of RECK were more marked, whereas the methylation-specific bands of RECK were almost undetectable (Fig. 1B).

To determine the effects of EGCG on the mRNA expression of RECK, RT-qPCR was performed to detect the mRNA expression levels of RECK in the SACC83 cells following treatment with a range of concentrations of EGCG for different durations (Fig. 2). The relative mRNA expression levels of RECK increased in a dose- and time-dependent manner (Fig. 2A and B).

As shown in Fig. 3, the protein expression levels of RECK were relatively low in the untreated SACC83 cells. Following treatment of the cells with different doses of EGCG for 6 days, and with 45 µm EGCG for different periods of time, the protein expression levels of RECK increased in the SACC83 cells (Fig. 3A and B).

The present study also investigated the effects of EGCG on cell invasion. As shown in Fig. 4, treatment with 5, 15 or 45 µm EGCG markedly suppressed the invasive ability of the SACC83 cells in a dose-dependent manner. These results suggested that restoration of the expression of RECK by EGCG is important for inhibiting the invasiveness of SACC83 cells.