Tumor and tumor-adjacent tissues samples of 45 patients with laryngeal carcinoma admitted to the People's Hospital of Xinjiang Uygur Autonomous Region (Xinjiang, China) from February 2009 to October 2013 were selected. These patients including 41 males and 4 females, the age of these patients ranged from 42-76 years old, with an average of 59.6±7.9 years. The data of overall survival (OS) rates of 45 cases were also collected from the hospital. The cut-off line of low and high expression of miR-490-5p were determined according to the median of its expression in these cases. The present study was approved by the Ethics Committee of the People's Hospital of Xinjiang Uygur Autonomous Region, and an informed consent was obtained from each patient.

To investigate the target gene of miR-490-5p, the biological information online analysis software of Targetscan (http://www.targetscan.org/vert_72/), miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/php/index.php) and miRDB (http://mirdb.org/miRDB/) were used.

NP69, BICR 18, FaDu, HNE-3 and Detroit 562 cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences. HaCaT cell lines (cat. no. 300493) were purchased from CLS Cell Lines Service GmbH. NP69 cell lines acted as the normal laryngeal cells, while HaCaT cell lines acted as normal tissue cells. The HNE-3, FaDu, NP69 cell lines were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.), whereas Detroit 562, BICR 18 and HaCaT cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.). The medium was supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.). Cells were cultured in an incubator filled with 5% CO₂ at 37°C and subcultured every 2-3 days. The overexpression plasmid of miR-490-5p (mimics, 5′-CCAUGGAUACUCCCCAGGCGGGU-3′) and MAP3K9 as well as their corresponding negative control (NC) plasmids were synthesized by Shanghai Genepharma Company and loaded into the pcDNA 3.1 plasmids. The transfection of all plasmids (50 nM) was conducted using Lipofectamine™ 2000 Transfection Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. Following transfection for 24 h, the efficiency of transfection was detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). For observing the effect of upregulated miR-490-5p on cell viability and predicting the target of miR-490-5p, BICR 18 and FaDu cells were treated with medium alone, negative control (NC) plasmid of miR-490-5p or the overexpression plasmid of miR-490-5p (mimics) and grouped respectively as control, NC and miR-490-5p mimics groups. The expression vector of MAP3K9 cDNA was transfected into BICR 18 and FaDu cells to achieve the effect of MAP3K9 overexpression. For determining the effectiveness of the overexpression of MAP3K9 in cells, BICR 18 and FaDu cells were treated with medium alone, NC plasmid of MAP3K9 or overexpression plasmid of MAP3K9-cDNA, which were respectively grouped as control, NC and MAP3K9 groups. For the investigation of the role of MAP3K9 in the regulation of miR-490-5p on cell viability, migration, invasion, EMT and colony formation, BICR 18 and FaDu cells were treated with medium alone, NC plasmid of miR-490-5p, overexpression plasmid of miR-490-5p (mimics), overexpression plasmid of miR-490-5p (mimics) and MAP3K9 as well as the overexpression plasmid of MAP3K9, which were grouped as control, NC, mimics, mimics + MAP3K9 and MAP3K9 group, respectively.

According to the manufacturer's protocol, total RNA of cells was extracted by TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNAs were synthesized by iScript™ cDNA Synthesis kit (Bio-Rad Laboratories, Inc.). The RT reaction was performed at 42°C for 30 min, followed by reverse transcriptase inactivation at 85°C for 15 sec. qPCR was performed using Fast Start Universal SYBR-Green Master kit (Roche Diagnostics) and the gene-specific primers. The reactions were run in 96-well plates using the following formula and program: 2X SYBR-Green master mix 10 μl, cDNA template 1 μl, forward primer (10 μM) 1 μl, reverse primer (10 μM) 1 μl, ddH₂O 7 μl, 2 min of hot start at 95°C 40 cycles for 15 sec at 95°C for 60 sec at 60°C and for 2 min at 10°C. The data were analyzed by the 2^-ΔΔCq method (18) with normalization to the control. Primers used are listed in Table I.

The assessment of cell viability was conducted using CCK-8 (HY-K0301; MedChemExpress). Cells in each group described above were seeded in a 96-well plate at a density of 10⁴-10⁵ cells/well in 100 μl of culture medium and incubated in an incubator with CO₂ at 37°C for 24 and 48 h. 10 μl of CCK-8 solution was added to each well of the plate, which was further incubated for another 1 h in the incubator. The absorbance of each sample was measured at 450 nm using a microplate reader (Multiskan; Thermo Fisher Scientific, Inc.) after the plate had been agitated on an orbital shaker for 1 min.

Cells were seeded in a 24-well plate at a cell density 6×10⁴ cells/well. The point mutation of MAP3K9 in BICR 18 and FaDu cells was created by the Quick-Change Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc.). Cells carrying wild or mutant type of MAP3K9 were grouped and treated as aforementioned. miR-490-5p was co-transfected with control vectors pRL-TK (Promega Corporation) coding for Renilla luciferase. Transfection was conducted using Lipofectamine™ 2000. Following transfection for 24 h, the luciferase activity was determined by GloMax 20/20 Luminometer (Promega Corporation). DNA sequences were confirmed by sequencing. Dual-Glo luciferase assay kit (Promega Corporation) was used to detect the luciferase activity after the cells had been gathered and lysed 48 h. Values of the firefly luciferase were normalized to Renilla.

Cells were lysed in 1 ml of radioimmunoprecipitation assay buffer (cat. no. 89900; Thermo Fisher Scientific, Inc.) and centrifuged at 4°C 16,000 × g for 30 min, and the supernatant of cells was then gathered. The concentration of total protein was determined by the Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). According to the protocol given by the manufacturer, 20 μl of standard proteins was diluted and 25 μl of samples were added into a 96-well plate. A total of 200 μl of the Working Regent was added to each well and well mixed for 30 sec. Plate was placed in the dark at 37°C for 30 min. The OD at 562 nm was read on a microplate reader (Multiskan; Thermo Fisher Scientific, Inc.).

Protein samples (20 μg/lane) were separated on 10% SDS-PAGE for 60 min at 80 V and transferred to a polyvinylidene difluoride membrane for 60 min at 80 V. The membrane was washed once in PBS with 0.2% Tween 20 (PBST) and then blocked with 5% non-fat milk for 1 h at room temperature (RT). The membrane was further probed with anti-MAP3K9 antibody (cat. no. ab228752), anti-β actin antibody (cat. no. ab115777), anti-matrix metalloproteinase (MMP)-9 antibody (cat. no. ab76003), anti-tissue inhibitor of metalloproteinase 1 (TIMP2) antibody (cat. no. ab180630), anti-epithelial (E)-Cadherin antibody (cat.no. ab40772), anti-vimentin antibody (cat. no. ab92547; all 1:1,000; Abcam) in non-fat milk and incubated overnight at 4°C. The membranes were then washed 3 times in PBST for 5 min and horseradish peroxidase-conjugated anti-rabbit immunoglobulin G secondary antibodies (cat. no. ab205718; Abcam) were added at 1:2,000 dilution in non-fat milk for 1 h at RT. The membranes were then exposed to Pierce™ enhanced chemiluminescence (ECL) plus western blotting substrate (Thermo Fisher Scientific, Inc.) for 1 min in the dark. The resulting bands were scanned and the images were captured in the ChemiDoc MP (Bio-Rad Laboratories Inc.) and the gray level of the bands was analyzed by the ImageJ software (version 1.46; National Institutes of Health).

Cells were seeded in the wells of a 6-well plate at a density of 5×10⁴ cells/well until 90% confluence was reached. Each confluent cell monolayer was scratched by a sterile plastic tip in order to produce a straight gap. The debris and the edge of the gap were washed with PBS. The cells were cultured in complete medium for 24 h and the relative wound distance was analyzed based on the images under the inverted light microscope (Olympus IX71; Olympus Corporation) at the same observation site. The distance between the gaps was set as the mean of the distance between upper, middle and lower edges. The distance in the images was measured by ImageJ software.

A total of 3×10⁴ cells with 100 μl serum-free medium were planted in the top chamber of a Transwell apparatus (8-μm) with a Matrigel-coated membrane (BD Biosciences) for Transwell invasion assay. The bottom chamber was filled with medium supplimented with 10% FBS. Following incubation of the Transwell for 12 h, the cells that invaded into the surface of the bottom chamber were fixed with 4% paraformaldehyde for 15 min at room temperature, stained with 0.5% crystal violet for 20 min at room temperature and counted under an Olympus IX71 microscope. The images of the bottom of each camber in the same site were imaged.

Cells were collected and seeded into the wells of a 6-well plate at a density of 200 cells/dish for 48 h. A total of 700 μg/ml of G418 (Abcam) was used to detect the positive cell clones for 3 weeks till the visible cell colony was visible. Medium was replaced every 3 days. Next, cell colony formations were gently washed with PBS and fixed with methanol for 15 min at room temperature. Colonies were stained with 0.5% crystal violet for 20 min at room temperature. The stain was then carefully washed off with running water and dried. Each colony in the wells was imaged and counted under an Olympus IX71 microscope.

All the data analysis was performed by GraphPad Prism v7.0 software (Graphpad Software, Inc.) and presented as mean ± standard deviation. The variantions between different groups were analyzed by one-way analysis of variance followed by Turkey's multiple comparison. The comparison of the OS rates was analyzed by log-rank test. P<0.05 was considered to indicate a statistically significant difference.

To confirm that the expression of miR-490-5p was downregulated in primary pharyngolaryngeal cancer tissues and cell lines as well as its effect on the prognosis, the mRNA level of miR-490-5p in tumor tissues, tumor-adjacent tissues, NP69, HaCaT, BICR 18, FaDu, HNE-3 and Detroit 562 cell lines was measured. As expected, the expression of miR-490-5p in tumor tissues was significantly decreased compared with in tumor-adjacent tissues (P<0.01; Fig. 1A). Similarly, the expression of miR-490-5p in BICR 18, FaDu, HNE-3 and Detroit 562 cells were decreased compared with HaCaT and NP69, particularly in BICR 18 and FaDu cells (P<0.05; Fig. 1C). Therefore, BICR 18 and FaDu cells were selected for later experiments. Furthermore, patients with low miR-490-5p expression demonstrated a significantly decreased OS rate compared with high miR-490-5p expression (P<0.05; Fig. 1B). The data from the present study suggested that the downregulation of miR-490-5p in pharyngolaryngeal cancer was a common condition, leading to an unfavorable prognosis.

In order to determine whether the upregulation of miR-490-5p could affect the cell viability of pharyngolaryngeal cancer cells, the miR-490-5p overexpression plasmid (mimics) was transfected into BICR 18 and FaDu cells, and the cell viabilities of BICR 18 and FaDu cells 24 and 48 h following culturing were measured. miR-490-5p was successfully overexpressed in BICR 18 and FaDu cells. The cell viabilities in BICR 18 and FaDu cells were significantly decreased in the mimic group compared with the NC groups following 48 h of culturing (P<0.05; Fig. 1D-G). The results demonstrated that the upregulation of miR-490-5p was able to lower the proliferation of pharyngolaryngeal cancer cells.

To understand how the upregulation of miR-490-5p led to decreased cell viability of BICR 18 and FaDu cells, the targets of miR-490-5p were predicted using targetscan, miRTarBase and miRDB databases and verified using a luciferase assay. The results of the Venn diagram demonstrated that TNKS2, MAP3K9, NUFIP2, CLCC1 and ABCC2 were target genes for miR-490-5p in results from targetscan, miRTarBase and miRDB databases (Fig. 2A). It was suggested that these five genes may be the target for miR-490-5p. Furthermore, the relative mRNA expression of MAP3K9 in miR-490-5p mimics group was significantly decreased compared with in NC groups in the BICR 18 and FaDu cells, while the other four genes exhibited no significant difference between the miR-490-5p mimics group and NC group (P<0.05; Fig. 2B and C). Moreover, the relative luciferase activities in the miR-490-5p mimics groups were significantly decreased compared with NC groups when the sequence of MAP3K9-3′-UTR was wild type, however, no significance difference was exhibited between NC and miR-490-5p mimics groups when the sequence of MAP3K9-3′-UTR was mutated in both BICR 18 and FaDu cells (P<0.05; Fig. 2D-F). Therefore, it was proved that MAP3K9 was the target gene for miR-490-5p. The expression of MAP3K9 in tumor tissues was significantly increased compared with in tumor-adjacent tissues (P<0.01; Fig. 2H), and the expression of MAP3K9 in tumor tissues and tumor-adjacent tissues was opposite to that of miR-490-5p (P<0.01; Fig. 2G and H).

In order to confirm that MAP3K9 was involved in the regulation of miR-490-5p on the cell proliferation, the expression of MAP3K9 was overexpressed in BICR 18 and FaDu cells, and further observed whether it could affect the cell viability in miR-490-5p-upregulated BICR 18 and FaDu cells. As demonstrated in the results of the present study, MAP3K9 was significantly overexpressed in BICR 18 and FaDu cells (P<0.05; Fig. 3A-D). Furthermore, the cell viabilities in miR-490-5p mimics + MAP3K9 groups were increased compared with the miR-490-5p mimics groups in both BICR 18 and FaDu cells, the cell viabilities in miR-490-5p mimics groups were decreased compared with in the NC groups in both BICR 18 and FaDu cells, while the cell viabilities in MAP3K9 groups were increased compared with in NC groups (P<0.05; Fig. 3E and F). Therefore, it indicated that miR-490-5p could target and inhibit the expression of MAP3K9 and lead to the downregulation of proliferation of pharyngolaryngeal cancer cells.

To confirm that MAP3K9 was involved in the regulation of miR-490-5p on the cell migration, the alteration of cell migration rate was measured by wound healing assay in BICR 18 and FaDu cells. The relative wound distances in miR-490-5p mimics groups were identified to be increased compared with in the NC groups but decreased in MAP3K9 groups compared with in NC groups in BICR 18 and FaDu cells (P<0.05; Fig. 4). Significantly, the cell viability in miR-490-5p mimics + MAP3K9 groups was decreased compared with in the miR-490-5p mimics groups in BICR 18 and FaDu cells (P<0.05; Fig. 4). The results of the present study suggested that miR-490-5p could target and inhibit the expression of MAP3K9, further leading to the downregulation of the migration of pharyngolaryngeal cancer cells.

To confirm that MAP3K9 was involved in the regulation of miR-490-5p on cell invasion, the change of cell invasion rate was measured by Transwell invasion assay in BICR 18 and FaDu cells. In the present study, the relative cell invasion rates (of control) in miR-490-5p mimics group were observed to be significantly decreased compared with in the NC groups in BICR 18 and FaDu cells and the relative cell invasion rates (of control) in MAP3K9 group was significantly increased compared with in NC groups in BICR 18 and FaDu cells (P<0.05; Fig. 5). Moreover, the relative cell invasion rates (of control) in miR-490-5p mimics + MAP3K9 groups were significantly increased compared with in the miR-490-5p mimics groups in BICR 18 and FaDu cells (P<0.05; Fig. 5). It suggested that miR-490-5p could target and inhibit the expression of MAP3K9 and further lead to the downregulation of the invasion of pharyngolaryngeal cancer cells.

To confirm that MAP3K9 was involved in the regulation of miR-490-5p on the migration, invasion and EMT process, the expression of MMP-9, TIMP-2, E-cadherin and vimentin was measured in BICR 18 and FaDu cells. It was demonstrated that the expression of MMP-9 and vimentin were significantly decreased, while the expression of TIMP-2 and E-cadherin were significantly increased in the miR-490-5p mimics group (P<0.05; Fig. 6). Compared with BICR 18 and FaDu cells in the NC groups, the expression of MMP-9 and vimentin were significantly increased, while the expression of TIMP-2 and E-cadherin were significantly decreased in MAP3K9 groups, compared with BICR 18 and FaDu cells in the NC groups (P<0.05; Fig. 6). In addition, the expression of MMP-9 and vimentin in miR-490-5p mimics + MAP3K9 groups were significantly increased compared with in the miR-490-5p mimics groups, while the expression of TIMP-2 and E-cadherin in miR-490-5p mimics + MAP3K9 groups were significantly decreased in BICR 18 and FaDu cells (P<0.05; Fig. 6) in the miR-490-5p mimics groups. The results of mRNA and protein expression levels were consistent, apart from the relative protein level of TIMP-2 in FaDu cells. It indicated that miR-490-5p could target and inhibit the expression of MAP3K9 and lead to the further downregulation of migration, invasion and EMT process of pharyngolaryngeal cancer cells.

To confirm that MAP3K9 was involved in the regulation of miR-490-5p on the ability of growth, the growth ability of BICR 18 and FaDu cells was measured by plate clone formation assay. The relative colony formation in the miR-490-5p mimics and MAP3K9 groups was significantly decreased and increased, respectively compared with in NC groups in BICR 18 and FaDu cells (P<0.05; Fig. 7). Furthermore, the relative colony formation in miR-490-5p mimics + MAP3K9 groups was significantly increased compared with the miR-490-5p mimics groups in BICR 18 and FaDu cells (Fig. 7; P<0.05). It suggested that miR-490-5p could target and inhibit the expression of MAP3K9 and lead to the downregulation of the ability of cloning of pharyngolaryngeal cancer cells.