The miRNA expression profiles of TGCA-HNSC dataset and the clinical information were obtained from TCGA database (https://portal.gdc.cancer.gov/) (25). A total of 528 patients with HNSCC from TCGA database were selected for analysis in the present study. The exclusion criteria were as follows: i) Incomplete information on LNM; ii) incomplete patient clinical data; and iii) the miR-411-5p expression data were incomplete. Following the exclusion of patients, the miR-411-5p expression levels and clinical information from 378 patients with HNSCC were used in the present study.

A total of 52 tissues from patients with HNSCC were collected from The Hospital of Stomatology, Sun Yat-Sen University (Guangzhou, China) between October 2017 and June 2018. No patient had received radiotherapy or chemotherapy before the biopsy, and all patients were diagnosed with HNSCC. The tissue samples were frozen at -80°C until required for use in further experiments. The histology and pathology of all samples was examined by two independent pathologists. Written informed consent was provided from all patients prior to participation and the study was approved by the Ethics Committee of Sun Yat-Sen University [approval no. ERC-(2017)-32].

The human HNSCC cell lines, HSC-3 and SCC-15, were purchased from the American Type Culture Collection. HSC-3 cells were cultured in DMEM supplemented with 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin. SCC-15 cells were cultured in DMEM/F12 (1:1) supplemented with 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin. All culture reagents were purchased from Gibco (Thermo Fisher Scientific, Inc.). All cells were maintained at 37°C in a humidified atmosphere with 5% CO₂.

A total of 1x10⁵ cells were transiently transfected with 50 nM chemically synthesized miR-411-5p mimic, 100 nM miR-411-5p inhibitor, 50 nM small interfering RNA (si)-RYBP and the corresponding negative controls (NCs; all Guangzhou RiboBio Co., Ltd.) using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. The sequences were as follows: miR-411-5p mimic, 5'-UAG UAG ACC GUA UAG CGU ACG-3'; miR-411-5p inhibitor, 5'-CGU ACG CUA UAC GGU CUA CUA-3'; and si-RYBP, 5'-ACA GCA TAC AGT CTG CAA A-3'. Lentiviruses encoding a reverse complementary sequence of miR-411-5p (Anti-miR-411-5p) and an empty vector (Anti-ctrl) were constructed by Shanghai GeneChem Co., Ltd. A total of 1x10⁵ cells HSC-3 and SCC-15 cells were infected with the lentiviral vectors (1x10⁸ TU/ml), and stable cell lines were selected following treatment with 2 μg/ml puromycin for 2 weeks.

Total RNA was extracted from tissues or cultured cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). To analyze the expression levels of miR-411-5p, Bulge-Loop™ miRNA qRT-PCR Primer sets were synthesized by Guangzhou RiboBio Co., Ltd. Total RNA was reverse transcribed into cDNA using the PrimeScript RT Master Mix (Takara Bio, Inc.), according to the manufacturer's protocols. qPCR was subsequently performed using the LightCycler® 480 SYBR Green I Master kit (Roche Diagnostics), according to the manufacturer's protocols. The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 5 min; followed by 40 cycles of denaturation for 10 sec at 95°C, annealing for 20 sec at 60°C and extension for 20 sec at 72°C; and a final 10 min extension at 72°C. The relative expression levels were calculated using the 2^-ΔΔCq method (26). The following primers were used for the qPCR: RYBP forward, 5'-GGG GTG GTG GGG TGG CAT ACT-3' and reverse, 5'-CGC AGA CGA AGG GTT TTG GGA TT-3'; and GAPDH forward, 5'-GCA CCG TCA AGG CTG AGA AC-3' and reverse, 5'-TGG TGA AGA CGC CAG TGG A-3'. GAPDH and U6 were used as the internal controls for RYBP and miR-411-5p, respectively. GAPDH and RYBP were purchased from Sangon Biotech Co., Ltd., while U6 and miR-411-5p were purchased from Guangzhou RiboBio Co., Ltd.

Cell migration and invasion assays were performed using Transwell chambers (8-mm pore size; Corning, Inc.). Briefly, for the migration assay, HSC-3 (3x10⁴) or SCC-15 (8x10⁴) cells were plated in serum-free medium into the upper chambers of the Transwell plates. For the invasion assay, HSC-3 (6x10⁴) or SCC-15 (2x10⁵) cells were seeded into the upper chambers with Transwell plates precoated with Matrigel for 6 h at 37°C. The lower chambers were filled with medium supplemented with 10% FBS. Following incubation for 24 h at 37°C, the medium was removed and the cells were fixed with 4% formaldehyde for 20 min at room temperature. Cells that had not migrated or invaded through the membrane were gently removed using a cotton swab. The fixed cells were subsequently stained with 0.4% crystal violet for 15 min at room temperature and observed under a light microscope (Zeiss GmbH). A total of five representative fields at x100 magnification were randomly imaged. ImageJ v1.8.0 (National Institutes of Health) was used for quantification, and the average counts of the five images represented the migrated cells.

The proliferation of HNSCC cells transfected with the miR-411-5p mimic or miR-411-5p inhibitor was determined using a CCK-8 assay (Telenbiotech). Briefly, 2x10³ cells were seeded into a 96-well plate and cultured for 24 h, prior to transfection with the miR-411-5p mimic or inhibitor. Following incubation for 24, 48, 72, 96 or 120 h, CCK-8 reagent was added to the culture medium for 2 h. The absorbance of each well was measured at a wavelength of 450 nm. All experiments were performed in triplicate.

For the colony formation assay, 5x10² cells were seeded into 6-well plates in triplicate 24 h post-transfection. Following the culture of cells for 10 days, the visible colonies were stained with 0.4% crystal violet for 10 min at room temperature. Colonies with diameters >1 mm were defined as colonies.

To further analyze the molecular mechanism of miR-411-5p-mediated metastasis in HNSCC, miRDB (http://www.mirdb.org/miRDB), miRTar-Base (http://mirtarbase.mbc.nctu.edu.tw/php/) and TargetScan 7.1 (http://www.targetscan.org/vert_71) databases were used. The potential miR-411-5p binding site in RYBP 3'-UTR was predicted using TargetScan (http://www.targetscan.org/vert_71). For luciferase assays, wild-type (Wt) or Mutant (Mut) RYBP 3'-UTR sequences were designed, synthesized and cloned into a pMIR-REPORT™ vector by OBiO Technology (Shanghai) Co., Ltd. to generate RYBP-3'-UTR-Wt and RYBP-3'-UTR-mut vectors, respectively. Then, HNSCC cells (4x10⁴) were co-transfected with 100 nM miR-411-5p mimics or NC and RYBP-3'-UTR-Wt or RYBP-3'-UTR-mut vectors using Lipofectamine 3000 reagent. Following 48 h of transfection, the relative luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega Corporation), according to the manufacturer's protocols, and was compared with the Renilla luciferase activity of the samples. The assays were performed in triplicate.

Total protein was extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with protease and phosphatase inhibitors. Total protein was quantified using a BCA kit (Wuhan Boster Biological Technology Co., Ltd.), according to the manufacturer's protocols, and 20 μg protein/lane was separated via SDS-PAGE on a 10% gel. The separated proteins were subsequently transferred onto PVDF membranes (EMD Millipore) and blocked with 5% non-fat milk for 1 h at room temperature. The membranes were then incubated with the following primary antibodies: Anti-E-cadherin (1:1,000; cat. no. 14472; Cell Signaling Technology, Inc.), anti-N-cadherin (1:1,000; cat. no. 13116; Cell Signaling Technology, Inc.), anti-vimentin (1:1,000; cat. no. 5741; Cell Signaling Technology, Inc.), anti-GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.) and anti-RYBP (1:500; cat. no. sc-374235; Santa Cruz Biotechnology, Inc.). Following the primary antibody incubation, the membrane was washed three times with TBS with 2% Tween-20 and incubated with HRP-conjugated anti-rabbit (cat. no. 7074) or anti-mouse (cat. no. 7076) secondary anti-bodies (1:5,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. Protein bands were visualized using Immobilon Western Chemiluminescent HRP substrate (EMD Millipore) and an ImageQuant™ LAS 4000 mini-imaging system (Cytiva). Semi-quantification was performed using ImageJ (v1.8.0).

IHC staining was performed with paraffin-embedded sections. Briefly, the lymph nodes were immediately fixed in 4% paraformaldehyde for 24 h and soaked in ethanol (70 to 100%), following which the sections (4 μm thick) were embedded in paraffin. The tissue sections were incubated in 10% goat serum blocking solution (Wuhan Boster Biological Technology, Ltd.) for 1 h at room temperature, followed by incubation with an anti-pan-cytokeratin primary antibody (1:200; cat. no. ab9377; Abcam) overnight at 4°C. Sections were then incubated with a secondary antibody for 30 min at room temperature according to the instructions of the GT Vision™ and Detection System/MoRb kit (Gene Tech), which contained the secondary antibody and DAB, followed by staining with DAB and counterstaining with hematoxylin for 1 min at room temperature. Then, two senior pathologists blinded to the data assessed and scored the IHC results. The extent of staining was scored according to the percentage of positively stained cells: i) 1, <10% of cells; ii) 2, 10-35% of cells; iii) 3, 35-70% of cells; and iv) 4, >70% of cells. The staining intensity was graded as follows: i) 0, no staining; ii) 1, weak staining (light yellow); iii) 2, moderate staining (yellow brown); and iv) 3, strong staining (brown), as previously described (9). The staining index was calculated by multiplying the percentage of positive cells and staining intensity. Five random fields per sample were analyzed using a light microscope (magnification, x100) to visualize sections.

All animal experiments were approved by the Ethical Committee of Sun Yat-Sen University (approval no. SYSU-IACUC-2020-000562) and were performed in accordance with the guidelines for animal care and protection (27). A total of 14 BALB/c nude mice (female; age, 4-6 weeks old; weight, 18-20 g) were obtained from GemPharmatech Co., Ltd., and were housed in specific pathogen-free conditions at 25°C and 50% humidity, with a 12-h light/dark cycle and free access to food and water. The mice were randomly divided into 2 groups (n=7). A total of 2x10⁶ SCC-15 cells with or without miR-411-5p expression knockdown were suspended in 100 μl PBS, and then injected into the tongue of the nude mice following anesthesia by isoflurane [4% (0.5 l/min) for induction and 2% for maintenance]. Following 8 weeks, the maximum tumor diameter in the mice was recorded as 5.65 mm and the maximum tumor volume was 62.40 mm³, all mice were sacrificed using cervical dislocation and death was confirmed by the absence of breathing, and the lack of a heartbeat and corneal reflex. Finally, the cervical lymph nodes were collected and analyzed using IHC analysis.

Statistical analysis was performed using SPSS version 20.0 software (IBM Corp.) and GraphPad Prism 6.0 software (GraphPad Software, Inc.). Statistical differences between two groups were determined using an unpaired Student's t-test, while multiple group comparisons were performed using a one-way ANOVA, followed by a Tukey's post hoc test. χ² tests were used to determine the association between miR-411-5p expression levels and clinicopathological features. Spearman's correlation analyses were used to determine the correlation between miR-411-5p and RYBP expression. The Kaplan-Meier method and a log-rank test were used for overall survival analysis. To further determine the influence of multiple independent prognostic factors, including miR-411-5p expression levels, on the overall survival of patients with HNSCC, multivariate Cox regression analysis was performed. P<0.05 was considered to indicate a statistically significant difference.

In the present study, 378 patients with HNSCC from TCGA were obtained, including 218 patients with LNM and 160 patients without LNM. As shown in Fig. 1A, the expression levels of miR-411-5p were upregulated in patients with HNSCC with LNM compared with patients without LNM. To validate the results obtained via bioinformatics analysis, miR-411-5p expression levels in 52 HNSCC clinical patient samples were analyzed by RT-qPCR, and the results revealed the same trends in the expression levels as the bioinformatics analysis (Fig. 1B). These findings suggested that the expression levels of miR-411-5p may be associated with LNM in HNSCC.

As shown in Table I, χ² analysis revealed that high miR-411-5p expression levels were positively associated with LNM, pathological differentiation and clinical stage in 52 clinical HNSCC patient samples. Furthermore, the association between miRNA-411-5p expression levels and the clinicopathological characteristics of the 378 patients with HNSCC are shown in Table SI. The analysis identified that miR-411-5p expression levels were associated with LNM; however, no association was found between miR-411-5p expression levels and sex, age, T stage, clinical stage and pathological differentiation. By combining the analysis of TCGA databases and HNSCC clinical samples, the data indicated that miR-411-5p expression levels may be a useful biomarker for HNSCC.

Kaplan-Meier survival analysis based on TCGA dataset revealed that upregulated miR-411-5p expression levels were associated with a poor prognosis in patients with HNSCC (Fig. 2A). In addition, the prognostic value of miR-411-5p expression levels was also analyzed in patients with LNM. The results revealed that upregulated miR-411-5p expression levels were associated with a poor overall survival rate in patients with HNSCC with LNM (Fig. 2B). Furthermore, univariate analysis revealed that LNM and miR-411-5p expression levels were prognostic factors for patients with HNSCC. Multivariate cox analysis of the prognostic variables in 378 patients with HNSCC from TCGA dataset identified that miR-411-5p expression levels [hazard ratio (HR), 1.478; 95% CI, 1.066-2.048; P=0.019] and LNM (HR, 1.911; 95% CI, 1.335-2.735; P<0.001) were significantly associated with the prognosis of HNSCC (Table SII). Taken together, these results suggested that the upregulated expression levels of miR-411-5p may be positively associated with the metastatic potential of HNSCC and may serve as a potential indicator for the poor prognosis of patients with HNSCC.

Clinicopathological analyses demonstrated that miR-411-5p expression levels were positively associated with LNM in HNSCC. Therefore, the effects of miR-411-5p on the migration and invasion of HNSCC cells were analyzed using Transwell assays. HSC-3 and SCC-15 cells were transfected with miR-411-5p mimics or inhibitors, compared with the negative control group, increased and decreased miR-411-5p expression levels in HNSCC cells was confirmed by RT-qPCR (Fig. 3A and B). Meanwhile, HSC-3 and SCC15 cells were infected with lentiviral vectors to establish stable cell lines that expressed short hairpin RNA against miR-411-5p (anti-miR-411-5p), decreased miR-411-5p expression was verified by RT-qPCR (Fig. S1A and B). In the miR-411-5p inhibitor group, the knockdown of miR-411-5p expression significantly reduced the migratory and invasive abilities of HSC-3 and SCC-15 cells (Fig. 3C). Conversely, the overexpression of miR-411-5p significantly promoted the migration and invasion of HSC-3 and SCC-15 cells (Fig. 3C). Furthermore, inguinal LNM models were constructed by injecting SCC-15 cells with or without knocked down miR-411-5p expression into the tongue of mice to analyze the effect of miR-411-5p on metastasis in vivo. Histological analysis revealed that the knockdown of miR-411-5p decreased the number of pan-cytokeratin-positive metastatic tumor cells and metastatic ratio of cervical lymph nodes (Fig. 3D and E). Taken together, these findings indicated that the knockdown of miR-411-5p may suppress LNM in vitro and in vivo.

It is well-established that the induction of EMT is a major event that promotes the motility of cancer cells in the invasion-metastasis cascade. Therefore, the effect of miR-411-5p on EMT was determined by analyzing the expression levels of EMT-related markers. The overexpression of miR-411-5p downregulated the expression levels of the epithelial marker, E-cadherin, and upregulated the expression levels of the mesenchymal markers, N-cadherin and vimentin, in HNSCC cells (Fig. 3F). By contrast, the genetic knockdown of miR-411-5p upregulated the expression levels of N-cadherin and vimentin and downregulated E-cadherin expression levels (Fig. 3F). These results indicated that miR-411-5p may promote the migration, invasion and EMT of HNSCC cells. Furthermore, CCK-8 and colony formation assays were conducted to analyze the proliferation of HNSCC cells. The results revealed that the overexpression and knockdown of miR-411-5p did not affect the proliferation of HNSCC cells (Fig. S2A and B).

The bioinformatics analysis demonstrated that the seed sequence of miR-411-5p was complementary to the 3'-UTR of RYBP (Fig. 4A), which suggested that RYBP may be a potential target of miR-411-5p. Dual-luciferase reporter assays confirmed that miR-411-5p overexpression significantly suppressed the relative luciferase activity of the RYBP-3'-UTR-Wt reporter gene, but had no effect on the RYBP-3'-UTR-Mut reporter gene (Fig. 4B). Moreover, RT-qPCR and western blotting confirmed that the overexpression of miR-411-5p significantly downregulated RYBP expression levels, while the silencing of miR-411-5p expression upregulated RYBP expression levels (Fig. 4C and D). Clinically, a negative correlation was identified between miR-411-5p and RYBP expression levels in HNSCC tissues (Fig. 4E). Collectively, these results suggested that RYBP may be a direct downstream target of miR-411-5p in HNSCC cells.

As expected, the expression levels of RYBP were downregulated in patients with HNSCC with LNM compared with patients without LNM (Fig. 5A). To analyze the effects of RYBP on HNSCC cells, si-RYBP was transfected into HSC-3 and SCC-15 cells. The genetic knockdown of RYBP resulted in the significant downregulation of RYBP protein expression levels (Fig. 5B). The results of the Transwell assays showed that the knockdown of RYBP significantly promoted HNSCC cell migration and invasion compared with the control siRNA (Fig. 5C and D). Furthermore, the knockdown of RYBP upregulated the expression levels of N-cadherin and vimentin, and downregulated E-cadherin expression levels in HSC-3 and SCC-15 cells (Fig. 5E and F).

To determine whether the knockdown of RYBP expression was able to rescue HNSCC cell migration and invasion in the anti-miR-411-5p HNSCC cell line, si-RYBP was transfected into the anti-miR-411-5p HNSCC cell line. The results of the Transwell assays revealed that the genetic silencing of RYBP in the anti-miR-411-5p HNSCC cell line increased cell migration and invasion compared with the NC group (Fig. 6A and B). Moreover, the silencing of RYBP rescued the miR-411-5p knockdown-induced downregulation of the expression levels of mesenchymal markers and upregulation of the expression levels of epithelial markers (Fig. 6C and D). Taken together, these results indicated that miR-411-5p may regulate HNSCC cell invasion, migration and EMT by targeting RYBP.