Patient recruitment and sample collection
A total of 22 patients with advanced HNSCC (21 men, 1 woman; mean age, 60.7 years) and 25 volunteers with no cancer (20 men, 5 women; mean age, 62.3 years) were investigated in this study (Table 1). Control patients had no history of cancers in the preceding 3 years. Peripheral blood samples were collected from patients with HNSCC who were receiving platinum-based chemoradiotherapy in the Department of Otorhinolaryngology-Head & Neck Surgery at Mie University Hospital, from January 2015 until December 2016. Blood samples were collected before initiating chemoradiotherapy. Within the patient group, we also compared plasma levels of miR-21 between the 15 patients with no recurrence and the 7 patients with recurrence. Clinical characteristics of the 22 patients, including age, sex, stage, tumor site, mean expression level of miR-21 in plasma, and Brinkman index (defined as [number of cigarettes per day] × [number of years during which the patient smoked]), CRP, neutrophil-lymphocyte ratio and SCC antigen are listed in Table 2. This study was approved by the ethics committee at Mie University Graduate School of Medicine (approval nos. 2445 and H2020-232). Written informed consent was obtained from each patient before enrolment in this study.
Cell culture
HNSCC FaDu and SAS cell lines (derived from human hypopharyngeal cell carcinoma and human tongue SCC, respectively) were obtained from RIKEN BRC Cell Bank (Tsukuba, Japan). These HNSCC cell lines were maintained in modified Eagle’s medium (MEM) medium or RPMI-1640 medium with 10% fetal bovine serum and antibiotics (1% penicillin, streptomycin), respectively. All human cell lines have been authenticated using short tandem repeat (STR) profiling this year.
RNA extraction from plasma
RNA was extracted from 200 µl of plasma by using an miRNeasy Serum/Plasma Kit (QIAGEN, Hiden, Germany) in the automated QIAcube (QIAGEN) as described previously [10], according to the manufacturer’s instructions. A synthetic Caenorhabditis elegans miR-39 miRNA mimic (QIAGEN) and carrier RNA (0.94 µg, MS2 bacteriophage total RNA; Roche Applied Sciences, Indianapolis, IN, USA) were spiked-in before RNA extraction. Isolated RNA was eluted in 15 µl of RNase-free water.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) for plasma miR-21
cDNA was synthesized from 5 µl of eluted RNA (containing miRNAs) in HiFlex buffer (QIAGEN), by using an miScript II RT Kit (QIAGEN) as described previously [10]. Next, quantitative RT-PCR was performed using the miScript SYBR Green PCR kit (QIAGEN) and miScript Primer Assays. MiRNA were normalized against the average of four reference miRNAs (miR-423-5p, miR-103a-3p, miR-191-5p, and miR-93), which were used as internal controls in plasma samples. In addition, syn-cel-miR-39 was used to confirm the extraction efficacy of RNA. All amplifications were carried out in an ABI Step One Plus Real-time PCR System (Applied Biosystems, Singapore, Singapore). Amplification curves were analyzed using SDS software version 2.2.2 (Applied Biosystems). Expression levels of mRNA were determined by using the 2−ΔΔC method.
Transfection experiments using miR-21 inhibitor
To transiently inhibit miR-21 expression, hsa-miR-21 mirVana™ miRNA inhibitor (Applied Biosystems) or hsa-miR-21 mirVana® mimics (Applied Biosystems) were used to transfect HNSCC cells, as described previously [18]. Verification of transfection efficiency was conducted using the mirVana™ miRNA Mimic Negative Control (Applied Biosystems) and mirVana™ miRNA Inhibitor Negative Control (Applied Biosystems), respectively. Forward transfections were conducted by mixing siRNA oligonucleotides (70 nM) with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) and Opti-MEM I (Invitrogen) and applying the mixture to cells at 24 h after plating. A series of in vitro assays was conducted after 48–72 h of incubation.
Cell viability assay
A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess the anti-proliferative effects of miR-21 inhibitor and proliferative effects of the miR-21 mimic on HNSCC cells, as previously described [19]. Cells were plated at a density of 4 ⋅103 cells/well into a 96-well plate, transfected with miR-21 mimic or inhibitor, and treated with either 48 h administration of cisplatin at various concentrations (0, 0.2, 1, or 5 µg/ml) or three exposures to radiation at various dose (0, 2, or 4 Gy), and incubated for a total of 72 h. Next, 10 µl of 5-mg/ml MTT (Sigma-Aldrich, St Louis, MO, USA) in 100 µl of medium was added to each well, followed by incubation for 4 h at 37°C. The medium was removed and replaced with 100 µl DMSO, and absorbance values were measured at 570 nm on a Bio-Rad model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
Cell apoptosis assay
Quantitative analysis of apoptotic and dead cells was performed by using An Annexin V and Dead Cell Assay Kit (Millipore, Burlington, MA, USA) with a flow cytometer (Muse™ Cell Analyzer; Millipore) as described previously [20], according to the instructions from the manufacturer. After incubating with cisplatin (0, 4, or 6 µg/ml) for 48 h or exposure to radiation (0, 4, or 12 Gy), all cells were harvested and diluted to a concentration of 5 ⋅105 cells/mL in MEM medium with 2% Fetal bovine serum (FBS). One hundred microliters of Annexin V and Dead Reagent and 100 µl of single cell suspension were then mixed in a microtube and analyzed by using the MuseTM Cell Analyzer (Millipore) after 30 min incubation in the dark at room temperature. All experiments were performed in quadruplicate.
Western blot analysis
After transfection with miR-21 mimic or inhibitor for 48 h, cells were harvested and lysed using RIPA buffer (Cell Signaling Technology, Dancers, MA, USA) supplemented with phenylmethylsulphonyl fluoride (Nacalai Tesque, Kyoto, Japan). Equal amounts of protein were runned by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (0.45 µm; Millipore). Membranes were washed with Tris-buffered saline (TBST) containing 0.1% Tween-20 (Nacalai Tesque) and 5% bovine serum albumin (Sigma-Aldrich, Saint Louis, MO, USA), and incubated overnight at 4°C with primary antibodies (PDCD4 antibody from Cell Signaling Technology, Danvers, MA, USA; β-actin antibody from MP Biomedicals, LLC, Irvine, CA, USA). After washing with TBST, membranes were further incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000; Santa Cruz Biotechnology, Dallas TX, USA) for 1 h at room temperature, and developed by using an electrochemiluminescence system in the end (GE Healthcare, Little Chalfont, UK). Protein bands were detected by an LAS4000mini imaging system (Fujifilm, Tokyo, Japan), and band intensities of Western blots were quantitatively measured by calculating integrated grayscale densities in consistently sized windows incorporating each band using ImageJ version 1.48 software as described previously [21] .
Statistical analysis
All statistical analyses were performed with SPSS version 23 statistical software. Two-group comparisons of ΔCt values between plasma samples from controls and HNSCC patients, as well as relapse-free and recurrent patients, were performed by using the Mann-Whitney U-test. Receiver operating characteristic (ROC) curves were chosen to evaluate the predictive power of miR-21 for tumor recurrence and to obtain the miR-21 cut-off values offering maximal sensitivity and specificity. The Kaplan-Meier method with log-rank comparison was selected to calculate OS, measured from the date chemoradiotherapy was initiated until the date of death from any cause (i.e., cancer-unrelated deaths were not censored), or last known follow-up for patients who were still alive. Statistical differences between groups were calculated by χ2 text, unpaired t-test, Mann-Whitney test, and one-way ANOVA with post-hoc multiple comparisons. Values of p<0.05 were considered to be a statistical significance.