Surgically resected OSCC specimens were obtained from 35 patients (aged 45 to 70, including 19 males and 16 females) with OSCC between June 2015 and June 2017 at Jining No. 1 People's Hospital (Jining, China) with written informed consent. According to the Union for International Cancer Control (UICC) standards in 2002, all the cases were classified as phases I–IV. In addition, individual oral tissue samples from 10 normal subjects (aged 35–65, including 6 males and 4 females) were collected as controls with written informed consent. The experimental procedures were approved by the Research Ethics Committee of Jining No. 1 People's Hospital.

H&E staining is a popular staining method in histology and it is one of the most widely used staining in medical diagnosis. The prepared 4-µm paraffin sections were incubated for 2 h in a 60°C incubator. The sections were deparaffinized by turpentine oil (TO) and sequentially soaked in 100, 95, 85 and 70% alcohol solutions for 2 min, and then hydrated using distilled water for 5 min. The sections were stained with hematoxylin for 5–15 min. Then, the excess dyeing solution on the slide was washed with water. Next, the sections were stained with eosin for 1–5 min. Subsequently, the sections were dehydrated using 70, 85, 95 and 100% alcohol, respectively, and were transparent through TO. Finally, the excess TO transparent agent around the section was wiped off, and a suitable amount of neutral resin was added, and the slide was covered.

Immunohistochemistry was used to detect PTEN expression in the OSCC specimens. The samples were fixed, embedded and cut into 4-µm thick sections. The sections were deparaffinized using xylene and rehydrated by increased grades of ethanol. A total of 0.5 µg primary rabbit polyclonal antibodies against PTEN (1:200; cat. no. 9188S; Cell Signaling Technology, Inc., Danvers, MA, USA) were added following antigen retrieval and incubated at 4°C overnight. Subsequently, immunohistochemical staining was performed using VECTASTAIN^® ABC immunohistochemistry kit (Cowin Biosciences, Co., Ltd., Beijing, China) according to the manufacturer's instructions.

ISH analysis was performed on deparaffinized 4-µm OSCC tissue sections using an ISH tissue implementation kit. OSCC sections were incubated at 60°C for 1 h and deparaffinized. Subsequently, the sections were rehydrated in decreasing concentrations of ethanol and washed in deionized water. The slides were incubated in 0.2 mol/l HCl for 5 min at room temperature and washed 3 times in phosphate-buffered saline (PBS) for 5 min. Proteins were digested for 15 min at 37°C following the addition of pepsin solution. Dehydration of sections was performed through incubation with 70, 95 and 100% ethanol for 1 min at each concentration. miR-21 probes labeled with digoxin were added to the hybrids. Sections were denatured at 90°C for 4 min, followed by incubation in ISH slide denaturation and hybridization solution at 37°C overnight. Post-incubation, coverslips and glue were removed and the slides were washed. Finally, the slides were observed under an inverted microscope.

The OSCC cell lines SCC15 and SCC25 were obtained from the American Type Culture Collection (ATTC; Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 100 µg/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified 5% CO₂ environment. miR-21 inhibitor and empty vector were chemically synthesized by Suzhou GenePharma Co., Ltd. (Suzhou, China) and transfected into SCC15 and SCC25 cells using Lipofectamine^™ RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.) according to the recommended protocol. Cells in which Lipofectamine™ RNAiMAX was only added were used as a control group. Both SCC15 and SCC25 Cells in each of the 3 groups (control, scramble and miR-21 inhibitor) were collected to be used for subsequent analysis at different time-points following transfection.

293T cells were obtained from ATTC and cultured in 24-well plates and co-transfected with 0.5 µg pMIR vectors containing PTEN 3′UTR or mutant (mut)PTEN 3′UTR and 20 µM miR-21 mimics or 20 µM miR-21 inhibitor using Lipofectamine^® RNAiMAX. Cells were lysed with Passive Lysis Buffer and collected at 48 h post-transfection. Luciferase activity was detected with a dual luciferase reporter assay kit according to the manufacturer's protocol. Renilla luciferase activity was used for normalization.

Total miRNAs and mRNAs were extracted from SCC15 and SCC25 cells using an miRNeasy Mini kit (Qiagen GmbH, Hilden, Germany) and Total RNA Purification kit (Qiagen GmbH) according to the manufacturer's instructions. Real-time RT-qPCR analysis was performed to validate miRNA and mRNA expression using a One-Step RT-PCR Kit. U6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Invitrogen; Thermo Fisher Scientific, Inc.) were used as endogenous controls. Each test was repeated in triplicate.

After transfection for 48 h, SCC15 and SCC25 cells were washed 3 times with cold PBS and lysed in a buffer composed of radio-immunoprecipitation assay buffer (Cowin Biosciences Co., Ltd.) and phenylmethanesulfonyl fluoride (Cowin Biosciences Co., Ltd.) (100:1). Total proteins were heated at 95°C for 10 min following measurement of protein concentration with an Enhanced BCA Protein Assay kit (Cowin Biosciences Co., Ltd.). Equal quantities (25 µg) of heated proteins from each sample were separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The blots were blocked in Tris-buffered saline with Tween-20 (TBST) containing 5% non-fat milk at room temperature for 1 h, then incubated with specific primary antibodies against against PTEN (1:1,000; cat. no. 9188S), phosphorylated (p)Akt (1:2,000; cat. no. 4060T), Akt (1:1,000; cat. no. 4691T) and GAPDH (1:1,000; cat. no. 5174S) (Cell Signaling Technology, Inc.) overnight at 4°C. Subsequently, the blots were rinsed 3 times with TBST and incubated with the secondary antibody for 1 h at room temperature. Protein bands were imaged using the AlphaView SA Western blot detection system (Carl Zeiss AG, Oberkochen, Germany) and quantified following normalization to the density of GAPDH using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Each test was repeated in triplicate.

Cell proliferation was assessed using Cell Counting Kit-8 (CCK-8; Cowin Biosciences Co., Ltd.) according to the manufacturer's instructions. Following treatment with miR-21 inhibitor, SCC15 and SCC25 cells were seeded into 96-well plates at a density of 4,000 cells/well and subsequently allowed to attach overnight. A 10 µl quantity of CCK-8 was added to each well at 0, 24, 48 and 72 h after transfection, and the cells were incubated for 1 h. Optical density (OD) was determined by spectrophotometric analysis at a wavelength of 450 nm. Each experiment was repeated in triplicate.

After transfection, SCC15 and SCC25 cells were transferred to 8-µm pore inserts and placed in companion wells containing DMEM and 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). The inserts were removed after a 12-h incubation, and the non-migrated cells on the upper surface were harvested. Cells on the lower surface were fixed with 5% glutaraldehyde (Beyotime Institute of Biotechnology, Shanghai, China) and stained with Giemsa (Beyotime Institute of Biotechnology) and counted under a fluorescent microscope. Each test was repeated in triplicate.

SCC15 and SCC25 cells were washed with PBS and resuspended in buffer at a concentration of 10⁶ cells/ml. Cells were mixed with 5 µl fluorescein isothiocyanate-conjugated Annexin V reagent and 5 µl propidium iodide (PI) (Invitrogen; Thermo Fisher Scientific, Inc.). At 15 min after incubation in the dark at room temperature, the samples were analyzed by flow cytometry (Beckman Coulter GmbH, Krefeld, Germany). Each test was repeated in triplicate.

All experiments were repeated at least 3 times and the data were presented as the mean ± standard deviation (SD). The results were analyzed by Student's t-test for the comparison of two and ANOVA (followed by Tukey's post hoc test) for the comparison of multiple samples using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant result.

The expression of miR-21 and PTEN was examined by ISH and immunohistochemical staining, respectively, in human OSCC tissues and normal tissues (Fig. 1). As depicted in Table I, miR-21 expression was observed in 80.0% (28/35) of OSCC tissues and in 30.0% (3/10) of normal tissues (P=0.003). By contrast, PTEN expression exhibited an opposite trend in OSCC tissues (37.1%, 13/35) and normal tissues (80.0%, 8/10; P=0.017) compared with the expression of miR-21. Furthermore, the association between miR-21 and PTEN expression and the clinicopathological profiles of patients was evaluated (Table II). The data indicated that the expression of miR-21 and PTEN was associated with tumor stage. miR-21 expression was lower in early stages (I+II) than in advanced stages (III+IV; P=0.028) and the expression of PTEN was significantly higher in early stages (I+II) than in advanced stages (III+IV; P=0.010). miR-21 and PTEN expression levels were not significantly associated with patient sex or age, tumor differentiation or lymph node metastases (P>0.05).

To knockdown endogenous miR-21, miR-21 inhibitor was synthesized and transfected into SCC15 and SCC25 cells. The expression of miR-21 and PTEN was then analyzed by RT-qPCR. The data indicated that miR-21 inhibitor efficiently silenced the expression of miR-21 compared with that in the control groups (Fig. 2A; P<0.01). By contrast, the gene expression of PTEN was notably upregulated in the miR-21 inhibitor group (Fig. 2B; P<0.01). No significant differences between the scramble and control groups were noted (P>0.05). The results indicated that miR-21 may be negatively associated with PTEN expression in SCC15 and SCC25 cells.

To investigate the molecular mechanism of miR-21 in the apoptosis of SCC15 and SCC25 cells, western blot analysis was performed following miR-21 silencing to assess the expression of PTEN, Akt and pAkt. The expression of PTEN was significantly increased (P<0.01) and the expression of pAkt decreased (P<0.05) in the miR-21 inhibitor groups compared with that in the control groups. There were no significant differences in Akt expression between the miR-21 inhibitor and control groups (P>0.05; Fig. 3). The results indicated that the level of PTEN expression in SCC15 and SCC25 cells was negatively regulated by miR-21.

The effect of miR-21 inhibitor on the proliferation and invasion of SCC15 and SCC25 cells was determined by CCK-8 and Transwell assays, respectively. As depicted in Fig. 4, both the proliferative and invasive abilities of SCC15 and SCC25 cells in the miR-21 inhibitor groups were significantly suppressed compared with those in the control groups (P<0.05). There were no significant differences between the control and scramble groups (P>0.05).

To detect the effect of miR-21 inhibitor on SCC15 and SCC25 cell apoptosis, Annexin V/PI analysis was performed. The data indicated that the percentage of apoptotic cells was markedly induced in the miR-21 inhibitor groups compared with that in the control groups (P<0.01; Fig. 5), indicating that miR-21 inhibitor transfection induced cell apoptosis.

To validate whether PTEN is a direct target of miR-21, PTEN 3′UTR and mutPTEN 3′UTR luciferase constructs were transfected into 293T cells with negative control (NC) mimics, miR-21 mimics, inhibitor NC or miR-21 inhibitor. Luciferase activity was assessed using a dual-luciferase reporter assay system. As illustrated in Fig. 6, compared with the cells in the other groups, the luciferase activity of 293T cells transfected with miR-21 mimics and PTEN 3′UTR was significantly reduced (P<0.05).