Oral squamous cell carcinoma (OSCC) is one of the most frequently occurring malignancies in the world. The RNA-binding protein quaking (QKI) is a newly identified tumour suppressor in multiple cancers, but its role in OSCC is currently unknown. The purpose of the present study was to clarify the relationship between QKI expression and OSCC development. We found QKI-5 expression to be significantly decreased in the oral cancer cell line CAL-27. QKI-5 overexpression also reduced the proliferation of CAL-27 cells, which correlated with cyclin D1. This regulative function of QKI-5 occurs by modulating the phosphorylation level of the mitogen-activated protein kinase (MAPK) pathway. Therefore this study shows that underexpression of tumour suppressor QKI-5 could activate the MAPK pathway and contribute to uncontrolled cyclin D1 expression, thus resulting in increased proliferation of oral cancer cells.
Oral squamous cell carcinoma (OSCC) is one of the most common malignancies in the world. Despite advances in its therapeutic management, OSCC has a very poor prognosis because of local recurrence and metastasis. However, the numerous genes that have been shown to affect the formation and development of OSCC imply various potential approaches to preventing and treating these cancers.
MicroRNAs and RNA-binding proteins are recognized as key regulators in cell biology, and function as post-transcriptional controllers in the process of mRNA splicing, stabilization, translation, and transportation. Defects in these molecules may result in various diseases, including cancers. As one of the evolutionarily conserved signal transduction and activation of RNA family members, the RNA-binding protein quaking 5 (QKI-5) contributes an alternative splicing function that targets a diverse set of tumourigenesis. In addition, recent findings indicate that genomic depletion of QKI-5 increases proliferation and dedifferentiation of cancer cells, which indicates that QKI-5 is a tumour suppressor for many cancer types.
However, no studies have examined the relationship between QKI-5 expression and OSCC, which could be of significance for improved diagnosis and treatment. In this study we determined, for the first time, that QKI-5 regulates the growth of human oral cancer cell line CAL-27, and that this regulative function occurs by binding to and deactivating the mitogen-activated protein kinase (MAPK) pathway, leading to decreased expression of cyclin D1. These findings imply that decreased QKI-5 may be a diagnostic and therapeutic target for OSCC.
Materials and methods
After surgical removal, all tissue specimens were immediately immersed into liquid nitrogen for 10 min and then stored at below −80 °C. Cancer tissues and adjacent normal mucosa tissues of every patient were reviewed and confirmed by the department of pathology of the study institution.
The in vitro expression construct pDsRed-N1-QKI-5 was generated by inserting the entire coding region of QKI-5 with a Kozak start sequence into the Bgl II/EcoR I sites of the expression vector pDsRed-N1. The primers for QKI-5 gene amplification were forward, 5′-GGGGATGGTCGGGGAAATG-3′ and reverse, 5′-GGGGTTAGTTGCCGGTGGCGGCTCG-3′. pBTM116 was used as a negative control for plasmid transfection.
Cell culture and transfection
Normal human oral keratinocytes (HOK) were purchased from ScienCell Research Laboratories and cultured according to the supplier’s instructions. Human oral cancer cell lines CAL-27, OECM1, and OC3 were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% foetal bovine serum (HyClone; GE Healthcare). Cells were maintained as monolayers in 60-cm 2 tissue culture treated flasks (Corning) at 37 °C in a humidified atmosphere of 5% CO 2 . Plasmid transfection was conducted using FuGENE 6 (Roche) in accordance with the manufacturer’s instructions.
Cells were seeded in 96-well plates (2 × 10 4 per well) and incubated for 24 h. Each experiment included blank controls (no plasmid), negative controls (transfected with pDsRed-N1 only), and samples with up-regulated QKI-5 (transfected with pDsRed-N1-QKI5). Cell viability was evaluated by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which is based on conversion of MTT to MTT–formazan by mitochondrial enzymes, as described previously.
Cell cycle analysis
The cell cycle was analyzed by flow cytometry. CAL-27 cells (2.5 × 10 6 ) transfected with pDsRed-N1-QKI5 or vector alone were trypsinized 24 h after transfection, fixed in 4% paraform, washed, and incubated in phosphate buffered saline containing propidium iodide (Sigma) for 30 min at 37 °C. Samples were then analyzed by fluorescence-activated cell sorting for DNA content.
Total RNA was extracted with TRIZol Reagent (Invitrogen) as per the manufacturer’s instructions. One microgram of total RNA was used as template for reverse transcription in accordance with the instructions of the M-MLV assay kit (Invitrogen). Primer sequences utilized in the study were the following: QKI-5 forward, 5′-ATCCTATTGAACCTAGTGGTGTA-3′, reverse, 5′-GGTCAGAAGGTCATAGGTTAGTT-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5′-CCCCGGTTTCTATAAATTGAGCC-3′, reverse, 5′-GTTTCTCTCCGCCCGTCTTC-3′. The real-time PCR reaction (Takara) was performed in a 10-μl system as per the manufacturer’s instructions. Results were analyzed using Bio-Rad CFX Manager software (Bio-Rad Laboratories).
Total cellular protein was isolated with radioimmunoprecipitation assay buffer (Cell Signaling), in accordance with the manufacturer’s instructions. A bicinchoninic acid assay was conducted to quantify the amount of protein, and 40 μg of protein for each sample was subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes; these were incubated overnight at 4 °C with primary antibodies against the following: QKI-5 (Sigma); cyclin A, D1, D2, and E (all from Santa Cruz Biotechnology, 1:1000); phospho-Akt, Akt, phospho-JNK, JNK, phospho-p38, p38, phospho-ERK1/2, ERK1/2, phospho-c-Jun, c-Jun, and GAPDH (all from Cell Signaling, 1:1000). Membranes were washed and then incubated with peroxidase-conjugated secondary antibody solution (KPL) for 1 h at 37 °C. Signals were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and gradation analysis was performed using GeneTools (Syngene).
The statistical analysis was performed using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). Statistical significance was evaluated by mean ± standard deviation, Student’s t -test, and one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant.
Decreased expression of QKI-5 in oral carcinoma
In order to investigate the role of QKI-5 in human OSCC development, we examined the expression of human OSCC tissues and adjacent normal mucosa tissues. The RT-PCR results showed that QKI-5 expression was significantly lower in tumour samples than in the adjacent normal mucosa tissues ( Fig. 1 A) . The QKI-5 expression level was also lower in the OSCC cell lines CAL-27, OECM1, and OC3 than in the HOK line ( Fig. 1 B). These data imply that QKI-5 down-regulation in oral cancer cells might be relevant to the development of OSCC.
QKI-5 inhibits cell cycle progression in oral cancer cell lines
To elucidate the biological function of QKI-5 in oral cancer, we transfected a QKI-5 expression vector into CAL-27 cells ( Fig. 2 A, B ) and studied their proliferation rate by MTT assay. Results showed that restoration of QKI-5 in CAL-27 significantly decreased proliferation 72 h after transfection ( Fig. 2 C). Flow cytometry results with propidium iodide staining showed the G 1 population to accumulate significantly in these transfected cells ( Fig. 2 D), which suggests that QKI-5 suppresses oral cancer by inhibiting cell cycle progression.
QKI-5 down-regulates cyclin D1
To characterize the molecular mechanisms by which QKI-5 arrests the cell cycle, we used real-time PCR to confirm its inhibitive effects on various cell cycle regulatory genes including cyclin A, cyclin D1, cyclin D2, cyclin E, CDK2, and CDK4. As shown in Fig. 3 A , the most notable change was the loss of cyclin D1 mRNA after QKI-5 expression. When normalized to GAPDH, there was a 61.6% reduction in cyclin D1 expression after QKI-5 restoration. The effects of QKI-5 on several cell cycle regulatory proteins were also examined ( Fig. 3 B). These data suggest that the QKI-5-mediated inhibition of proliferation is primarily due to cyclin D1 down-regulation.