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Introduction

Hypopharyngeal cancer is a common malignant tumor, which has a poor prognosis among head and neck cancer (1,2). Hypopharyngeal carcinoma originates in the mucosal epithelia in the hypopharynx (3–5). Hypopharyngeal cancer is invasive, yet the majority of patients exhibit a lack of evident early symptoms (6). In addition, the hypopharynx is not part of routine medical exams, thus the majority of patients with hypopharyngeal cancer exhibit advanced disease at diagnosis (7); the 5-year survival rate is <20% for patients with advanced disease (8). Surgery, chemotherapy and radiotherapy are used in combination in the clinical treatment of hypopharyngeal cancer; however, outcomes of these treatments are not satisfactory (9,10). Therefore, the development of novel strategies and effective methods to treat hypopharyngeal cancer is imperative.

Alternative splicing has a powerful role in regulating gene expression and increasing protein diversity (11,12). Alternative splicing is performed by heterogeneous nuclear ribonucleoprotein and splicing factor proteins (13–15). RNA-binding motif protein 17 (RBM17), which is a part of the RNA spliceosome complex (16), binds to the single-stranded three AG dinucleotides at the exon/intron border, and acts in the second catalytic step of mRNA splicing (17). The N-terminal domain of RBM17 contains a G-patch that has been implicated in an interaction between proteins and protein/nucleic acid (18,19), and the C-terminal domain contains an RNA recognition motif for mRNA splicing (17). RBM17 is also involved in DNA repair (20). Expression of RBM17 is low in normal tissues, including those of the breast, liver and prostate; however, its overexpression has been found in a number of solid tumor types including breast, pancreas and prostate cancer (21). However, the role of RMP17 in hypopharyngeal carcinoma remains unclear.

The present study investigated the effects of RBM17-knockdown on cell proliferation, cell cycle and apoptosis in the hypopharyngeal carcinoma cell line FaDu using lentivirus-mediated specific shRNA targeting RBM17.

Materials and methods

Cell culture

FaDu cells were purchased from Cell Bank of Chinese Academy of Science (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 µg/ml streptomycin (Sangon Biotech Co. Ltd, Shanghai, China). Cells were maintained in an incubator with 5% CO2 and 95% humidity at 37°C. FaDu cells were infected with the lentiviral vectors containing small interfering RNAs (siRNAs) targeting RBM17 or empty vectors.

Lentiviral construction for shRNA treatment

RBM17-specific shRNA (5′-ACTTAAGTGTCCTACTAAA-3′; GenBank NM_032905) and the negative control sequence (5′-AATTCTCCGAACGTGTCACGT-3′) were cloned into AgeI and EcoRI sites of the pGV115-green fluorescent protein (GFP) lentiviral vector (Shanghai Genechem Co., Ltd., Shanghai, China). The plasmids used were pGV115-GFP-shRBM17 for specific interference of RBM17 and pGV115-GFP-negative control (NC) for control. Inhibition of RBM17 expression in FaDu cells with RBM17-specific shRNA was performed as follows. Lentivirus was generated in FaDu cells as described previously (22). Briefly, FaDu (2×105 cells/well) cells were seeded into 6-well plates; when cell growth reached ~80% confluency, appropriate volumes of lentiviral vectors were transfected into FaDu cells for 48–72 h to generate lentivirus. Lentivirus was harvested and the viral titer was measured with a Centricon-plus-20 (EMD Millipore, Billerica, MA, USA). The cells were used in subsequent experiments when the rate of infected cells reached 70% at 72 h post-infection.

Human FaDu cells (2×105 cells/well) were reseeded into 6-well plates and incubated with either a RBM17-shRNA (1×106 TU) or control-payload lentiviruses (1×106 TU) for 8–12 h. A total of 72 h post-infection, infected FaDu cells were observed under a fluorescent imaging microscope (Olympus Corporation, Tokyo, Japan) at ×100 magnification by counting green cells based on GFP intensity. The efficiency of this infection was determined by RT-PCR and western blotting, which were performed according to the subsequent steps.

Cell proliferation assay

FaDu cells were infected with RBM17 shRNA lentivirus (shRBM17) or non-silencing shRNA lentivirus, and 2×103 cells were seeded with 100 µl medium/well into 96-well plates. Cell growth and viability was evaluated on days 1, 2, 3, 4 and 5. For cell growth, FaDu cells at the logarithmic phase after being infected with either the shCtrl or shRBM17 lentivirus and the plates were counted using the Cellomics ArrayScan™ VT1 automated reader (Cellomics, Inc.; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for each day. In each well, ≥800 cells were analyzed. Each experiment was performed in triplicates. For cell viability, at the given time, 20 µl MTT (5 mg/ml; Sangon Biotech Co., Ltd.) was added into each well and plates were incubated for 4 h at 37°C. The medium in each well was then removed and crystals were dissolved by the addition of 150 µl dimethyl sulfoxide (Sangon Biotech Co., Ltd.) in each well. Following a 10 min incubation at room temperature, the absorbance was measured at 570 nm.

Reverse transcription-quantitative polymerase chain reaction

Total RNA was isolated from the FaDu cell lines using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was used in a reverse transcription reaction to synthesize cDNA according to the manufacturer's protocol using RevertAid® First Strand cDNA Synthesis Kit (MBI Fermantas; Thermo Fisher Scientific, Inc.). PCR primers were designed by Beacon Designer 7 software (Premier Biosoft International, Palo Alto, CA, USA). Primer sequences were as follows: RBM17 forward, 5′-TCAAATCCGCTGACTGAAATAC-3′ and reverse, 5′-ACCTCCCATTCAAGTCAACAA-3′; and GAPDH forward, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′. Quantitative PCR was performed according to Takara SYBR® Master Mix kit instructions (Takara Biotechnology Co., Ltd, Dalian, China) as following: 95°C for 15 sec, followed by 45 cycles of 95°C for 5 sec and 60°C for 20 sec. The 2−ΔΔCq method was used to analyze relative changes in gene expression (23).

Flow cytometry analysis of cell cycle distribution

For cell cycle analysis, cells infected with RBM17-shRNA lentivirus or NC lentivirus were seeded in 6-well plates and cultured at 37°C for 5 days prior to analysis. Cells were collected by centrifugation at 1,200 × g for 5 min at 4°C, washed twice with ice-cold PBS, fixed with cold 70% ethanol for 1 h at 4°C, and stained with 50 µg/ml propidium iodide (PI) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in the presence of 100 µg/ml RNase (Sangon Biotech Co., Ltd.) at 37°C for 30 min. Cells were analyzed by flow cytometry using a FACSCalibur™ flow cytometer (BD Biosciences, San Diego, CA, USA), according to the manufacturer's protocol. Data was analyzed using FlowJo Software (version 10; FlowJo LLC, Ashland, OR, USA).

Analysis of apoptosis by flow cytometry

To analyze apoptosis, cells were stained with binding buffer containing annexin V-allophycocyanin (cat. no. 88-8007; eBioscience, San Diego, CA, USA) at 25°C in the dark for 10 min. Cells were analyzed by flow cytometry using a FACSCalibur flow cytometry (BD Biosciences) according to the manufacturer's protocol. Data was analyzed using FlowJo Software (version 10; FlowJo LLC).

Western blot analysis

FaDu cells were lysed and homogenized on ice in lysis buffer (cat. no. C500001; Sangon Biotech). Homogenates were centrifuged at 12,000 × g for 20 min at 4°C. The protein concentration of each sample was measured using Modified BCA Protein Assay Kit (cat. no. C503051; Sangon Biotech). Equal volumes (~20 µg total soluble proteins of supernatants) were separated on 12% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes. Membranes were incubated with rabbit polyclonal antibody specific for RBM17 (1:500; cat. no. 101441; Abcam, Cambridge, UK) and monoclonal antibody specific for GAPDH (1:2,000, cat. no. sc-32233; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. Membranes were then rinsed 3 times and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:10,000, cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Membranes were visualized using an EasyBlot ECL kit (Bio Basic Inc., Markham, ON, Canada).

Statistical analyses

Data were expressed as mean ± standard deviation. Student's t-test was performed to analyze differences between two groups using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). P<0.05 were considered to indicate a statistically significant difference.

Results

RBM17 mRNA and protein is knocked down in cells infected with RBM17-shRNA

A high infection efficiency was observed by measuring GFP expression via fluorescence microscopy (Fig. 1A). Expression of RBM17 mRNA in cells infected with RBM17-siRNA was significantly decreased compared with control cells (P<0.01; Fig. 1B). Western blot analysis revealed that RBM17 protein levels were reduced in cells infected with RBM17-siRNA compared with control cells (P<0.01; Fig. 1C).

Figure 1. Expression of RBM17-knockdown in hypopharyngeal carcinoma cell line FaDu. (A) Representative images of cells in culture taken 3 days post-infection using light and fluorescent microscopy are shown. Original magnification, ×100. (B) At 5 days post-infection, RBM17 mRNA expression was significantly reduced in FaDu RBM17-shRNA knockdown cells as compared with FaDu shCtrl cells (**P<0.01). (C) RBM17 protein expression analyzed by western blot analysis in shCtrl and shRBM17 groups. GAPDH was used as the internal control. RBM17, RNA binding motif protein 17; shCtrl, control short hairpin RNA.

Knockdown of RBM17 inhibits growth of hypopharyngeal carcinoma cells

To analyze the effect of regulation of RBM17 levels on the proliferation of hypopharyngeal carcinoma cells, the aforementioned FaDu cells infected with lentivirus were analyzed by Cellomics every day for 5 days. Results from day 4 and 5 of the assay revealed that proliferation of FaDu cells was significantly inhibited (P<0.01; Fig. 2), indicating that knockdown of RBM17 reduces the proliferative ability of hypopharyngeal carcinoma cells.

Figure 2. Effect of RBM17 expression on proliferation of hypopharyngeal carcinoma cells. (A) Images of cell growth (initial magnification, ×100). (B) Human hypopharyngeal carcinoma cell growth was assayed every day for 5 days. (C) The cell growth rate was monitored on days 1, 2, 3, 4 and 5 by assay. shCtrl vs. shRBM17, **P<0.01. RBM17, RNA binding motif protein 17; shCtrl, control short hairpin RNA; OD, optical density.

Downregulation of RBM17 increases the proportion of human hypopharyngeal carcinoma cells in G2/M phase

To demonstrate the effects of RBM17-knockdown on the cell cycle of hypopharyngeal carcinoma cells, a flow cytometry assay was performed. FaDu cells transfected with RBM17-specific shRNA had a higher proportion of cells in G2/M phase than did the control cells (P<0.01; Fig. 3).

Figure 3. Effects of RBM17-knockdown on the cell cycle. (A) Cell cycle analysis of FaDu cells was determined by flow cytometry. (B) The proportion of different cell cycle phases. The proportion of cells in the G2/M phase is significantly increased in cells infected with RBM17-siRNA (**P<0.01). RBM17, RNA binding motif protein 17; siRNA, small interfering RNA.

Knockdown of RBM17 in human hypopharyngeal carcinoma cells increases apoptosis

To observe the interplay between RBM17 and apoptosis, apoptosis was measured by flow cytometry in FaDu cells in which RBM17 was knocked down (Fig. 4A). The proportion of apoptotic FaDu cells was significantly increased in shRBM17 cells compared with control cells (P<0.01; Fig. 4B), indicating that RBM17 expression is a determinant of apoptosis in human hypopharyngeal carcinoma cells.

Figure 4. Effects of RBM17-shRNA transfection on cell apoptosis. (A) Cell death was determined by annexin V staining and flow cytometry. (B) Apoptosis was increased in cells infected with RBM17-shRNA compared with control cells (**P<0.01). RBM17, RNA binding motif protein 17; siRNA, small interfering RNA.

Discussion

RBM17 is part of the spliceosome complex, and is involved in the alternate splicing of mRNA with AGs at the exon/intron border (16). The expression of RBM17 is limited in the majority of normal tissues, and is high in epithelial cells and a number of types of cancer tissue (21). However, the biological function of RBM17 is poorly understood.

RBM17 has been confirmed to be overexpressed in multiple cancer types (21,24). A recent study revealed that overexpression of RBM17 suppresses cell proliferation and adhesion to fibronectin (25). Furthermore, the overexpression of RBM17 is known to induce cell migration and invasion (26). To investigate RBM17 function in the hypopharyngeal carcinoma FaDu cell line, RBM17 expression was knocked down in FaDu cells. The proliferation of FaDu cells infected with RBM17-shRNA was reduced, and downregulation of RBM17 increased apoptosis in FaDu cells and the proportion of cells in G2/M phase. These findings indicate that RBM17 accelerates the growth rate of FaDu cells.

The results of the present study imply that RBM17 may be associated with cell cycle checkpoints responsible for maintaining genomic integrity and regulating cellular proliferation in FaDu cells (27). The G2 checkpoint responds to DNA damage, with cancer cells with DNA damage passing through the S phase checkpoint but remaining at the G2 phase checkpoint (28). A previous study found that RBM17 was involved in DNA repair (20). However, an increase in the proportion of cells infected with RBM17-shRNA in G2/M phase in the present study indicates the association of RBM17 with the decreased DNA repair.

In summary, the present study underlines the potential roles of RBM17 in the human hypopharyngeal carcinoma FaDu cell line. The results of the current study indicate that knockdown of RBM17 by shRNA reduces the proliferation of FaDu cells, and the knockdown of RBM17 increased the proportion of cells undergoing apoptosis and arrested the cell cycle at the G2/M phase. These findings provide the basis for further investigation of the precise mechanism by which RBM17 influences cell biology in hypopharyngeal carcinoma.

Acknowledgements

The present study was supported by the Key Project of Anhui Provincial Department of Education (grant no. KJ2015A284).

Competing interests

The authors declare that they have no competing interests.

References