The human cSCC cell line A-431 and the 293-T cell line were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The cells were maintained in a humidified incubator with 5% CO₂ at 37°C.

A total of 15 paired tumor and adjacent normal tissues (5 cm away from each tumor) were obtained at the same locations from patients diagnosed with cSCC during surgery between August 2015 and March 2017 at the First Affiliated Hospital of Jinan University (Guangzhou, China). These cSCC cases included 8 male patients and 7 female patients with a mean age of 62.7 years (range, 38–87 years). All tissue samples were collected, immersed in liquid nitrogen and maintained at −80°C for further experiments. Written informed consent was obtained from each patient prior to the current study. The study protocol was approved by the Research Ethics Committee at the First Affiliated Hospital of Jinan University (Guangzhou, China).

For the overexpression and knockdown experiments of miR-186, mimic (3′-CAAAGAAUUCUCCUUUUGGGCU-5′), inhibitor (3′-AGCCCAAAAGGAGAAGGCUUUG-5′) and negative control (NC; 3′-UUCUCCGAACGUGUCACGUTT-5′) sequences were designed by Shanghai GenePharma Co., Ltd. (Shanghai, China). A-431 cells were transfected with the miRNA NC, miR-186 mimic or miR-186 inhibitor (all 100 nM) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according the manufacturer's protocol for 24 h.

For APAF1 knockdown, APAF1 siRNA (si-APAF1; 3′-GUGCCUACAAAGGUGUUAUUU-5′) and NC siRNA for APAF1 (NC-siRNA; 3′-UUCUCCGAACGUGUCACGUUU-5′) were designed by Shanghai GenePharma Co., Ltd. The A-431 cells were transfected with si-APAF1 or NC-siRNA (both 25 nM) with or without miR-186 inhibitor (100 nM) using Lipofectamine 2000 according to the manufacturer's protocol for 24 h.

Total RNA was extracted from cSCC and control tissues, and miR-NC-, miR-186 mimic- or miR-186 inhibitor-transfected A-431 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). mRNA was reverse transcribed into cDNA using the Revert Aid First Strand cDNA Synthesis kit (cat. no. K1622; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, and the thermocycling conditions were 25°C for 5 min, 42°C for 60 min and 70°C for 10 min. qPCR was performed using SYBR-Green Real-Time Master mix (Toyobo Life Science, Osaka, Japan) following the manufacturer's protocol. The thermocycling conditions of qPCR were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. GAPDH and U6 were used as internal controls for APAF1 and miR-186, respectively. The primers used for the detection of miR-186 and APAF1 were as follows: miR-186, sense 5′-GCGGCGCAAAGAATTCTCCT-3′ and antisense 5′-GTGCAGGGTCCGAGGT-3′; APAF1, sense 5′-ATGGACACCTTCTTGGACGACAG-3′ and antisense 5′-TGTGGGGGCGGACAACTAA-3′; GAPDH, sense 5′-TGTTCGTCATGGGTGTGAAC-3′ and antisense 5′-ATGGCATGGACTGTGGTCAT-3′; U6, sense 5′-CGCTTCGGCAGCACATATACTA-3′ and antisense 5′-CGCTTCACGAATTTGCGTGTCA-3′. Primers were designed by Sangon Biotech Co., Ltd. (Shanghai, China). The relative expression of miR-186 and APAF1 were calculated and normalized using the 2^−ΔΔCq method (25).

Total protein was extracted from the tissue samples and miR-NC-, miR-186 mimic-, miR-186 inhibitor, si-APAF1+miR-186 inhibitor or NC-siRNA+miR-186 inhibitor-transfected A-431 cells using an SDS lysis buffer (cat. no. P0013G; Beyotime Institute of Biotechnology, Haimen, China) on ice for 30 min. The concentration of total protein was measured using a bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). A total of 50 µg/lane protein was separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 10% low fat dried milk at 25°C for a minimum of 1 h, followed by incubation with primary antibodies against APAF1 (1:1,000; cat. no. ab32372), light chain 3B (LC3-B; 1:500; cat. no. ab48394) or Beclin1 (1:1,000; cat. no. ab62557; all Abcam, Cambridge, MA, USA) for 1 h at room temperature (RT). Membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (cat. no. BA1054; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 2 h at RT. GAPDH was used as the internal control and signals were detected using enhanced chemiluminescent reagents (cat. no. SW2030; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The band net optical density was analysed using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA).

The TargetScan database (targetscan.org) predicted one binding site for miR-186 on APAF1. A wild-type (WT) APAF1 [WT-3′-untranslated region (UTR)-APAF1] with the predicted miR-186 target binding sequence and a mutant APAF1 (MUT-3′-UTR-APAF1) with a mutation in the binding site, were synthesized and cloned into the psi-CHECK2 vector (cat. no. C8021; Promega Corporation, Madison, WI, USA). The 293-T cells were seeded in 96-well plates at a density of 1×10⁴ cells/well. Following overnight incubation at 37°C, the 293-T cells were transfected with the reconstructed plasmid containing WT-3′-UTR-APAF1 or MUT-3′-UTR-APAF1 in the presence of miR-186 mimics or the negative control (NC) by Lipofectamine^® 2000. Cells were harvested and the luciferase activity was measured using a Dual-Luciferase Reporter assay system (Promega Corporation, Madison, WI, USA) 48 h following transfection. Renilla luciferase activity was used for normalization of the firefly luciferase activity.

miR-NC-, miR-186 mimic- or miR-186 inhibitor-transfected A-431 cells were seeded at a density of 1×10⁵ cells/ml on a coverslip pre-coated with poly-L-lysine. They were subsequently fixed with cold 4% formaldehyde at 4°C overnight. After washing three times with PBS containing 0.1% Triton X-100, cells were blocked with 10% bovine serum albumin (cat. no. FA016-50G; Amresco, LLC, Solon, OH, USA) for 2 h at RT followed by incubation with primary antibodies against APAF1 (1:1,000; cat. no. ab2001; Abcam) and DAPI (1:2,000; cat. no. ab104139; Abcam) at 4°C overnight. Cells were then incubated with Alexa Fluor 488 donkey anti-mouse immunoglobulin G (1:200; ab150105; Abcam) secondary antibodies for 1 h at RT and visualized using a confocal laser-scanning microscope. Magnification at ×200.

si-APAF1+miR-186 inhibitor or NC-siRNA+miR-186 inhibitor-transfected A-431 cells were seeded into 6-well plates and incubated overnight at 37°C. Cells were fixed with 50 µl cold 4% formaldehyde for 30 min at RT. Then the cells were washed twice with cold PBS. Hoechst 33258 was added to the wells at a concentration of 20 µg/ml (Sigma-Aldrich; Merck KGaA) and incubated for a minimum of 20 min at RT. Following washing with PBS, the cells were visualized using a Leica confocal laser-scanning microscope (TCS SP8; Leica Microsystems GmbH, Wetzlar, Germany) at 365 nm. Magnification at ×400.

The Click-iT Plus EdU Alexa Fluor 1594 Imaging kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used according to the manufacturer's protocol, to determine the effects of miR-186 mimics or inhibitor on cell proliferation. miR-NC-, miR-186 mimic- or miR-186 inhibitor-transfected A-431 cells were fixed with 50 µl cold 4% formaldehyde for 30 min at RT. DAPI (1:2,000) was used to stain the cell nucleus for 30 min at RT and signals were detected using an Olympus FLUOVIEW FV1000 confocal laser-scanning microscope (Olympus Corporation, Tokyo, Japan) at a magnification of ×100.

A-431 cells transfected with miR-186 NC, mimic or inhibitor were seeded onto glass dishes at a density of 1×10³ cells/ml and incubated in an atmosphere containing 5% CO₂ at 37°C for 2 weeks. The cells were fixed with 50 µl cold 4% formaldehyde for 30 min at RT and subsequently stained with 0.1% crystal violet for 15 min at RT. Local cloning morphology was photographed with an inverted microscope. The colonies were counted and each of the experimental conditions was performed by using a Nikon Eclipse Ti inverted microscope (Nikon Corporation, Tokyo, Japan) in triplicate. Magnification at ×100.

To evaluate the effects of miR-186 on the invasive ability of cSCC cells, a Matrigel assay was performed. miR-NC-, miR-186 mimic-, miR-186 inhibitor, si-APAF1+miR-186 inhibitor or NC-siRNA+miR-186 inhibitor-transfected A-431 cells were suspended in 100 µl DMEM at a concentration of 1×10⁵ cells/ml and seeded into the upper Transwell chamber with an 8-µm pore size coated with Matrigel (Corning Inc., Corning, NY, USA). A total of 200 µl DMEM containing 15% FBS was added to the lower Transwell chamber and cells were cultured at 37°C for 24 h. Cells in the upper chamber were removed and those in the lower chamber were fixed with 4% paraformaldehyde for 30 min at RT stained with 0.1% crystal violet for 5 min at RT and observed using a Nikon Eclipse Ti inverted microscope in triplicate Magnification at ×200.

A wound-healing assay was used to assess the effects of miR-186 on cell migration ability. miR-NC-, miR-186 mimic- or miR-186 inhibitor-transfected A-431 cells were seeded in 6-well plates at a concentration of 1×10⁴ cells/well. A parallel wound was made using a pipette tip once the A-431 cells reached 100% confluence. Cells were then cultured at 37°C in a 5% CO₂ atmosphere and images were captured using an inverted microscope at 0 and 48 h. Magnification at ×100.

To assess cell apoptosis and perform cell cycle analysis, miR-NC-, miR-186 mimic- or miR-186 inhibitor-transfected A-431 cells were seeded in 24-well plates at a density of 1×10⁵ cells/well and cultured in DMEM with 10% FBS at 37°C for 24 h. For the cell cycle distribution analysis, the cells were digested by trypsin (Gibco; Thermo Fisher Scientific, Inc.), washed with PBS three times and fixed with 80% ethanol for 5 min at 4°C. They were then incubated with 0.25 mg/ml Ribonuclease A (Sigma-Aldrich; Merck KGaA) for 30 min at 37°C and 20 µg/ml propidium iodide (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) for 20 min at room temperature. For the cell apoptosis analysis, the cells were digested with trypsin, centrifuged at 111.8 × g for 5 min at 4°C, washed with PBS, re-suspended in 100 µl 1X binding buffer [cat. no. 70-AP101-100-BB; Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd., Hangzhou, China]. Then the cells were double stained with an Annexin V-FITC/PI apoptosis detection kit (Bestbio Company, Shanghai China) in the dark for 15 min at room temperature. Following staining, the apoptosis rate and cell cycle distribution was analyzed using a BD Accuri™ C6 Plus flow cytometer and equipped with CellQuest software (version 6.1×; both BD Bioscience, San Jose, CA, USA) according to the manufacturer's protocol.

Data are presented as the mean ± standard deviation and all statistical analyses were performed using SPSS 20.0 software (IBM Corp., Armonk, NY, USA). Cell experiments had >3 biological replicates. One-way analysis of variance and the least significant difference post hoc multiple comparison test were used to compare the differences among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

To investigate the precise roles of miR-186 and APAF1 in the tumorigenesis of cSCC, their expression in cSCC tissues was compared with corresponding normal tissues. RT-qPCR results revealed that miR-186 expression was significantly higher in cSCC tissues compared with the corresponding normal tissues (P<0.001, Fig. 1A). Conversely, APAF1 expression was significantly reduced in cSCC tissues compared with the control samples (P<0.001, Fig. 1B). In addition, the relative expression of APAF1 was negatively correlated with the expression of miR-186 in cSCC tissues (R=−0.665, P<0.01; Fig. 1C). Western blotting revealed that the expression of APAF1 protein was reduced in cSCC tissues compared with the controls (Fig. 1D). These results suggest that miR-186 and APAF1 may serve a critical role in the pathogenesis of cSCC.

To further explore the correlation between APAF1 and miR-186, the TargetScan miRNA target predication database was used. One predictive target site for miR-186 was identified in APAF1 (Fig. 2A). A dual-luciferase reporter assay revealed that the relative luciferase activity was significantly attenuated by miR-186 mimics in the WT-3′-UTR-APAF1 system, whereas it was not significantly altered by the application of miR-186 mimics in the MUT-3′-UTR-APAF1 system (P<0.001; Fig. 2B). These results suggest that the APAF1 gene is a direct target of miR-186.

To determine how miR-186 regulates the expression of APAF1, RT-qPCR, immunohistochemistry and western blotting were performed to detect APAF1 expression in A-431 cells with miR-186 overexpression or knockdown. The RT-qPCR results revealed that miR-186 expression was significantly increased in A-431 cells transfected with miR-186 mimics and significantly decreased in A-431 cells transfected with miR-186 inhibitors compared with the NC group (P<0.001 and P<0.05, respectively; Fig. 3A). The RT-qPCR assay also demonstrated that APAF1 expression was significantly downregulated in A-431 cells transfected with miR-186 mimics and significantly upregulated in those transfected with miR-186 inhibitors (P<0.01; Fig. 3B). Immunohistochemistry and western blotting also supported these results (Fig. 3C and D). Western blotting indicated that the expression of LC3-B and Beclin1 was lower in A-431 cells transfected with miR-186 mimics and markedly higher in A-431 cells transfected with miR-186 inhibitors compared with the NC group (Fig. 3D). These results indicate that APAF1 expression is directly regulated by miR-186 in the A-431 cSCC cell line.

To understand the role of miR-186 in the tumorigenesis of cSCC, proliferation, invasion, migration and apoptosis were investigated in A-431 cells transfected with miR-186 mimics or inhibitors. EdU staining and colony formation assays were used to examine the effects of miR-186 on cell proliferation. The results revealed the proliferation ability was increased in A-431 cells transfected with miR-186 mimics compared with the NC-transfected cells, while proliferation was decreased in A-431 cells transfected with miR-186 inhibitors (Fig. 4A and B). Transwell and wound-healing assays demonstrated that invasion and migration were significantly enhanced in A-431 cells transfected with miR-186 mimics and significantly attenuated in A-431 cells transfected with the miR-186 inhibitor compared with the NC-treated cells (P<0.01; Fig. 4C and D). Cell apoptosis was not significantly affected in A-431 cells transfected with miR-186 mimics compared with the NC group; however, it was significantly upregulated in A-431 cells transfected with miR-186 inhibitors (P<0.01; Fig. 4E). Flow cytometry was performed to assess the effects of miR-186 on cell cycle distribution. The results revealed that miR-186 overexpression significantly decreased the percentage of A-431 cells in the G0/G1 phase and significantly increased the percentage of A-431 cells in S phase compared with the NC group (P<0.05 and P<0.01, Fig. 4F). These results suggest that miR-186 may act as an oncogene in the cSCC A-431 cell line.

To further explore the role of APAF1 in cSCC, miR-186 expression was assessed using RT-qPCR and the expression of APAF1, LC3-B and Beclin1 proteins was analyzed using western blotting in A-431 cells transfected with miR-186 inhibitors with or without si-APAF1. The results revealed that miR-186 expression was significantly decreased in the si-APAF1 transfected group compared with the control group (P<0.01; Fig. 5A). Western blotting demonstrated that APAF1, LC3-B and Beclin1 protein expression was notably decreased in A-431 cells transfected with miR-186 inhibitors and si-APAF1, compared with the A-431 cells transfected with miR-186 inhibitors alone (Fig. 5B). A Matrigel assay and Hoechst staining were performed to evaluate the effect of APAF1 on cell invasion and apoptosis in A-431 cells transfected with miR-186 inhibitors. The results suggest that cell invasion is significantly enhanced, while migration is markedly attenuated in A-431 cells transfected with miR-186 inhibitors and si-APAF1 compared with A-431 cells transfected with the miR-186 inhibitor alone (P<0.01; Fig. 5C and D). These results suggest that APAF1 may act as a tumor suppressor in the A-431 cSCC cell line.