Twenty-three pairs of primary HSCC and corresponding normal epithelial samples were obtained from patients with HSCC at Chiba University Hospital (Chiba, Japan) from 2005 to 2013. The samples considered normal were free of cancer cells as determined by pathologic examination. The backgrounds and clinicopathological characteristics of the patient are summarized in Table I. The patients were classified according to the 2002 Union for International Cancer Control (UICC) TNM staging criteria prior to treatment. Written consent for tissue donation for research purposes was obtained from each patient before tissue collection. The protocol was approved by the Institutional Review Board of Chiba University. The specimens were immersed in RNAlater (Qiagen, Valencia, CA, USA) and stored at −20°C until RNA was extracted.

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA concentrations were determined spectrophotometrically, and molecular integrity was checked by gel electrophoresis. RNA quality was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

The following human cell lines were used: FaDu (derived from a primary lesion of hypopharyngeal SCC), SAS (derived from a primary lesion of tongue SCC), HSC3 (derived from lymph node metastasis of tongue SCC), HCT116 p53^+/+ (derived from a colon adenocarcinoma) and its p53^−/− derivative. All cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO₂ at 37°C.

cDNA synthesis and PCR procedures were described in our previous reports (13,27). The expression levels of miR-504 (Assay ID: 002084) were analyzed by TaqMan quantitative real-time PCR (TaqMan^® MicroRNA Assay; Applied Biosystems) and normalized to RNU48 (Assay ID: 001006). TaqMan® probes and primers for CDK6 (P/N: Hs01026371_m1), RB1 (P/N: Hs01078066_m1), CDC23 (P/N: 00946641_m1), CCND1 (P/N: 00765553_m1) and GUSB (P/N: Hs00939627_m1) as an internal control were obtained from Applied Biosystems (Assay-On-Demand Gene Expression Products). The ΔΔCt method was adopted and applied to calculate the relative quantities of subject genes. All reactions were performed in triplicate and included negative control reactions that lacked cDNA.

To perform gain of function studies, we utilized two different sources of mature miR-504: miR-504-A, Ambion Pre-miR miRNA Precursor, PM12429 (Applied Biosystems) and miR-504-T, miRIDIAN Mimic, MIMAT0002875 (Thermo Scientific Dharmacon, Waltham, MA, USA). Pre-miR Negative Control #2 (AM17111, Applied Biosystems) was used for negative control experiments. The miRNA transfection procedures and confirmation of miRNA transfection efficiency were described in our previous reports (13,27).

Cells were transfected with 10 nM miRNA by reverse transfection and plated in 96-well plates at 3×10³ cells per well. After 72 h, cell proliferation was determined with the XTT assay using the Cell Proliferation Kit II (Roche Molecular Biochemicals, Mannheim, Germany) as previously reported (13,27).

Cell cycle status was examined using an APC BrdU Flow kit (BD Bioscience, San Jose, CA, USA) according to the manufacturer’s protocol. Briefly, SAS and FaDu cells were transiently transfected with miR-control, miR-504-A or miR-504-T. Seventy-two hours after transfection, 10 μM BrdU was added to the medium and incubated for 6 h. The cells were then trypsinized and fixed with paraformaldehyde and permeabilized with saponin. After DNase treatment, the cells were stained with anti-BrdU antibodies and 7AAD and analyzed with a FACSCalibur flow cytometer (BD Bioscience).

To identify miR-504 target genes, we used genome-wide gene expression analysis and in silico analysis. First, we performed genome-wide gene expression analysis using miR-504 transfection of SAS and FaDu. SurePrint G3 Human GE 8×60K Microarray (Agilent Technologies) was used for expression profiling of miR-504 transfectants in comparison with negative control miRNA transfectants. The genes that were downregulated in miR-504 transfectant were then categorized into Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using GeneCodis analysis (28) (http://genecodis.cnb.csic.es/). The target site for miR-504 was analyzed with TargetScan Release 6.2 (http://www.targetscan.org/).

Cells were harvested 72 h after transfection and lysates were prepared. Protein (50 μg) from each lysate was separated on a Mini-Protean TGX gel (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes. Immunoblotting was performed with mouse CDK6 antibody (1:500, #3136, Cell Signaling Technology, Danvers, MA, USA) with GAPDH antibody (1:1,000, ab8245, Abcam, Cambridge, UK) used as an internal control. The membrane was washed and incubated with anti-mouse IgG HRP-linked antibody (#7076, Cell Signaling Technology). Complexes were visualized with an Immun-Star™ Western Chemiluminescence kit (Bio-Rad), and the expression levels of these genes were evaluated by ImageJ software (ver.1.44; http://rsbweb.nih.gov/ij/).

The relationships between two groups and the numerical values obtained by qPCR were analysed using the paired t-test. Spearman’s rank test was used to evaluate the correlation between the expression of miR-504 and target genes. The relationships among more than three variables and numerical values were analysed using the Bonferroni adjusted Mann-Whitney U test. All analyses were performed using Expert StatView (version 4, SAS Institute Inc., Cary, NC, USA).

To validate our past miRNA profiling results, we evaluated miR-504 expression in 23 clinical HSCC specimens. The expression levels of miR-504 were significantly lower in tumor tissues than in corresponding adjacent normal epithelia (miR-504 expression normalized to RNU48: normal, 0.0026±0.0011; tumor, 0.00070±0.00052, P<0.0001, Fig. 1). The expression levels of miR-504 in HNSCC cell lines were also lower than those in normal epithelia (miR-504 expression normalized to RNU48: FaDu, 0.000026; SAS, 0.00016; HSC3, 0.000025; Fig. 1).

To investigate the role of miR-504, we performed gain-of-function studies using mature miRNA transfection of three HNSCC cell lines (FaDu, SAS and HSC3). We utilized two sources of mature miR-504 (miR-504-A, Ambion; miR-504-T, Thermo Scientific Dharmacon) to ensure reproducibility of the data.

The XTT assay demonstrated that cell proliferation was significantly inhibited in miR-504 transfectants in comparison with the mock or miR-control transfectant cells. Specifically, we observed the following growth, expressed as a percentage of the mock: i) FaDu-mock, 100.0±1.0; miR control, 92.6±2.7; miR-504-A, 45.5±2.3; miR-504-T, 76.4±1.9; ii) SAS-mock, 100.0±6.8; miR control, 92.0±3.9; miR-504-A, 34.0±2.4; miR-504-T, 45.1±3.3; iii) HSC3-mock, 100.0±3.7; miR control, 103.4±3.9; miR-504-A, 81.6±2.7; miR-504-T, 89.6±4.9, with P<0.0083 (Fig. 2).

Because miR-504 has been reported to promote tumorigenicity by regulating TP53, we evaluated functional effects of miR-504 in HCT116 p53^+/+ cells and its p53^−/− derivative cell line. The XTT assay showed that cell proliferation was significantly inhibited by miR-504 transfection in both cell lines, suggesting that miR-504 functioned as a tumor suppressor regardless of p53 status. We observed the following growth, expressed as a percentage of the mock: i) HCT116 p53^+/+-mock, 100.0±3.7; miR control, 94.5±3.8; miR-504-A, 68.7±2.2; miR-504-T, 74.9±3.8; ii) HCT116 p53^−/−-mock, 100.0±4.4; miR control, 82.8±2.9; miR-504-A, 63.8±2.1; miR-504-T, 65.9±2.1, with P<0.0083 (Fig. 2).

To study whether miR-504 affected the cell cycle status of cancer cells, we performed flow cytometric analysis of cells stained with anti-BrdU and 7AAD allowing the discrimination of cell fractions that resided in G0/G1, S or G2/M phases of the cell cycle (Fig. 3A). The fraction of FaDu cells in the G0/G1 phase was significantly larger in miR-504 transfectants in comparison with the miR control transfectants, whereas the fraction of SAS cells in G2/M phase was significantly larger in miR-504 transfectants (Fig. 3B).

To identify miR-504 target genes, we used genome-wide gene expression analysis and in silico analysis. First, we performed genome-wide gene expression analysis using two cancer cell lines (FaDu and SAS) and selected genes downregulated by miR-504 transfection compared with miR control transfection. In this analysis, 810 genes and 1,145 genes were recognized as downregulated genes (log₂ ratio <−0.5) in FaDu and SAS, respectively. Entries from the microarray data were approved by the Gene Expression Omnibus (GEO) and were assigned GEO accession no. GSE37119.

Next, genes downregulated in miR-504-transfectants were categorized into KEGG pathways using GeneCodis analysis and 24 pathways were identified as significantly enriched in both lines (Tables II and III). Among these pathways, we focused on the ‘cell cycle’ pathway because this pathway has been implicated in cancer cell proliferation. A total of 19 genes were identified in this pathway and four genes (RB1, CDK6, CDC23 and CCND1) had putative miR-504 target sites predicted by the TargetScan database (Table IV).

We investigated the expression levels of four candidate genes in HSCC clinical specimens. CDK6 was significantly upregulated in cancer tissues (P=0.0004, Fig. 4A). Furthermore, the expression of CDK6 was inversely correlated with that of miR-504 in HSCC specimens (r=−0.43, P=0.0039, Fig. 4B).

We performed qPCR and western blotting in FaDu and SAS to investigate whether CDK6 expression was downregulated by restoration of miR-504. CDK6 mRNA expression was significantly repressed by miR-504 transfection of FaDu cells, while no changes were observed in SAS cells (Fig. 5A). The expression levels of CDK6 protein were repressed in miR-504 transfectants in comparison with mock or miR-control transfectants in both FaDu and SAS cells (Fig. 5B).