Participants
The OSCC tissues and corresponding normal tissues (>2 cm from the edge of the tumor) were collected from 72 patients who underwent surgical resection in the Department of Stomatology, the Fourth Affiliated Hospital of Hebei Medical University between January 2015 and December 2016. The patients had complete data and definite diagnosis by pathological examination and were all first episode patients. The subjects had no treatment for OSCC before the operation and signed written informed consent. The samples were collected from 35 males and 37 females with a mean age of 53 years (range, 21–75 years). The Ethics Committee of the Fourth Affiliated Hospital (Shijiazhuang, China) approved the experiment and supervised and guided the whole experimental process (approval no. 201411EC040). All patients were followed up immediately after discharge. By 31st December 2019, 49 cases survived and 23 cases succumbed to the disease.
Materials and reagents
The following materials were used to investigate the expression of the p53 gene: primers of MEG3, p53, pcDNA3.1-MEG3 overexpression plasmids and an empty control plasmid (Wanleibio Co., Ltd.). The normal oral mucosa cell line (hNOK) and OSCC cell line SCC25 and CAL27 were purchased from BeNa Culture Collection; Beijing Beina Chunglian Institute of Biotechnology). Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco; Thermo Fisher Scientific, Inc. BSA, DMSO and MTT reagent were purchased from Sigma-Aldrich; The expression of the p53 gene was detected using TRIzol® RNA isolation kit, Lipofectamine® 2000 liposome, Matrigel, Invitrogen Superscript Reverse Transcriptase kit and SYBR Green qPCR Master Mix kit which were all purchased from Invitrogen (Thermo Fisher Scientific, Inc.). Rabbit anti-human monoclonal and goat anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology Co., Ltd.
Reverse transcription-quantitative (RT-q)PCR experiment
The total RNA was extracted from the appropriate tissues using TRIzol for RNA quality inspection, and cDNA was prepared via reverse transcription. Fluorescence quantitative analysis was performed according to the operational instructions of the SYBR Green qPCR Master Mix kit. According to the target sequence, the corresponding upstream and downstream primers were designed and synthesized for PCR amplification. GAPDH was used as internal reference gene. The primer sequences were as follows: lncRNA MEG3 forward, 5′-CTTTTCTGGGGGAATGGGG-3′ and reverse, 5′-AGAGGGGTGGGAAGGGACT-3′; p53 forward, 5′-ACCACCATCCACTACAACTACAT-3′ and reverse, 5′-CAGGACAGGCACAAACACG-3′; and GADPH forward 5′-CTTAGTTGCGTTACACCCTTTCTTG-3′ and reverse, 5′-CTGTCACCTTCACCGTTCCAGTTT-3′. The thermocycling conditions were as follows: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec, elongation at 72°C for 30 sec and final extension at 72°C for 2 min 30 sec. The expression of lncRNA was calculated with GAPDH as internal reference. After three independent experiments, the data were obtained by using the following formula: ∆Cq=Cq lncRNA-Cq GAPDH; and ∆∆Cq=(Cq lncRNA-Cq GAPDH)-(Cq lncRNA-Cq GAPDH) experimental group-(Cq lncRNA-Cq GAPDH) control group. When the relative expression of OSCC and normal tissues were counted, the relative expression of each sample (2−∆∆Cq) and the changes in the lncRNA in different tissues were compared (37).
Cell culture and plasmid transfection
The SCC25 and CAL27 cells were seeded in DMEM containing 10% FBS and cultured in a 95% CO2 incubator at 37°C. The medium was changed, and subculturing was performed every 3–4 days. The cells in the logarithmic growth phase were seeded on a 6-well plate and transfected when the cells grew to ~70% confluency. The PEGFP-N1 plasmid (Clontech; Takara Bio USA) transfection was performed according to the manufacturer's instructions of the Lipofectamine 2000 transfection reagent. The transfection concentration was 40 nmol/l at 37°C for 20 min. After transfection, the cells were cultured in the incubator for 6 h, and then the medium was replaced with DMEM containing 10% FBS. After 48 h of transient transfection, the RNA was extracted to test the transfection efficiency.
Experimental grouping
The experiment was divided into the following: blank control group (NC), untransfected SCC25 and CAL27 cells; vector group (empty vector), SCC25 and CAL27 cells transfected with pcDNA3.1 empty control plasmid; and overexpression group (pcDNA3.1-MEG3), SCC25 and CAL27 cells transfected with pcDNA3.1-MEG3.
MTT assay
The SCC25 and CAL27 cells were seeded on 96-well plates with 1×104 cells in each well. After the cells adhered to the wall, transfection was performed. Each group was set with 3 multiple wells. After 6, 24, 48, 72 and 96 h of culture, 10 µl of MTT solution was added to each well, and the incubation was continued for at 37°C for 4 h. DMSO was used for purple formazan dissolution. The OD value was measured at 570 nm by a microplate analyzer, and the cell growth curve was plotted (38).
Transwell chamber assay
For the migration experiment, the experimental groups were the same as those in section. After 48 h, the transfected cells were collected, and the cells were suspended in serum-free medium at a density of 2.5×105 cells/ml. Then, 200 and 600 µl of the serum-free medium were added to the upper and lower chambers of 6.5 mm Transwell with 8.0 µm pore polycarbonate membrane insert (Corning, Inc.), respectively, which was stored at 37°C for 1 h. The medium in the well was absorbed and discarded, and the chamber was washed twice with PBS. Then, 200 µl of the cell suspension was added in the upper chamber, and 800 µl of the medium containing 30% FBS was added to the lower chamber cultured at 37°C for 48 h. The Transwell chamber was cleaned with PBS, fixed with 4% paraformaldehyde at 25°C for 20 min and stained with 0.5% crystal violet for 5 min. The cells migrated to the lower layer of the microporous membrane were counted under an inverted microscope (magnification, ×200). Five visual fields/samples were counted, and the mean was determined. For the invasion assay, Matrigel gel was thawed at 4°C and diluted with serum-free medium at the ratio 1:3 under a clean worktable. The Transwell chambers were placed in 24-well plates coated with 40 µl of Matrigel and then incubated in an incubator at 37°C for 2 h. The remaining steps were the same as those listed for the migration experiment.
Flow cytometric analysis
The cells were inoculated in 6-well plates with 5×105 cells per well. After 48 h of transfection, the cells in each group were collected and washed twice with PBS, and 500 µl of the binding buffer was added to gently suspend the cells. Annexin VLIGHT 650/PI apoptosis detection kit was purchased from Wanleibio Co., Ltd., which was used according to the manufacturer's protocols. Annexin V-light 650 (5 µl) was added, and the mixture was evenly mixed. Then, 10 µl of propidium iodide was added, and the mixture was evenly mixed again. The cells were incubated at room temperature for 15 min, and apoptosis was detected via flow cytometry (using Beckman Analysis 2.0) and analyzed using Kaluza Analysis 2.0 (Beckman Coulter, Inc.).
Western blot analysis
The protein was extracted using total protein extraction kit (Wanleibio Co., Ltd.). The protein concentrations were detected using bicinchoninic acid protein quantification kit (Wanleibio Co., Ltd.). After 48 h of transfection, the p53 protein was detected using a Bio-Rad vertical electrophoresis system. Then, 5% of concentration gel and 12% of separation gel were selected as electrophoresis plastic. Next, 5X Loading Buffer and PBS were used to dilute the protein sample, which was boiled in a water bath for 5 min, and 20 µl solution was prepared with 40 µg protein loaded per lane. The proteins were transferred to PVDF blotting membrane at 80V for 1.5 h, and 5% (M/V) skimmed milk powder with TBST (0.05% Tween20) buffer was used for standby. After the transfer, the PVDF membrane was removed, immersed in TBST in the shaker at speed of 100 rpm for 5 min at 25°C. Then, TBST buffer was removed and the PVDF membrane was immersed in skimmed milk powder solution in the shaker at speed of 100 r/min for 1 h. The transferred proteins were probed with respective primary antibodies against b-actin (1:1,000; cat. no. WL01845, Wanleibio Co., Ltd.) and P53 (1:500, cat. no. WL01333, Wanleibio Co., Ltd.) overnight at 4°C. After the membrane was washed with TBST buffer, the horseradish peroxidase-labeled secondary antibody (1:5,000, cat. no. WLA023, Wanleibio Co., Ltd.) was diluted with 5% skimmed milk powder/TBST solution to the relevant concentration. Then, the membrane was added into the second antibody solution, and the signals were developed using an enhanced chemiluminescent kit (cat. no. WLA003, Wanleibio Co., Ltd.) and was prepared for color development. The images were collected, exposed and the captured using Gel-Pro Analyzer 4.0 (Media Cybernetics, Inc.) system (39).
Statistical analysis
SPSS 22.0 software (IBM Corp.) was used for statistical analysis. The measurement data was expressed as mean ± SD. The measurement data was normally distributed, and the square difference was the same. The significance of the average values was analyzed using one-way ANOVA with a Tukey's HSD post hoc test. χ2-test and Fisher's Exact test were performed to analyze the count data. P<0.05 was considered to indicate a statistically significant difference.