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Introduction

Tongue squamous cell carcinoma (TSCC) is one of the most frequent types of oral carcinoma, and is characterized by a high growth and metastatic potential (1,2). TSCC usually causes dysfunctions in speech, mastication and deglutition (2). Currently, the predominant treatment options available to patients with TSCC are surgery, radiotherapy and chemotherapy (3). Despite the improvements in its diagnosis and treatment, the prognosis of TSCC has not improved in recent decades, probably due to its late diagnosis, as ~50% of patients are diagnosed at stages III and IV (4,5). Previous studies have demonstrated that oncogene activation and tumor suppressor gene inactivation may be implicated in the pathogenesis of TSCC (6,7). However, the detailed molecular mechanisms underlying TSCC development and progression have yet to be elucidated (8). Therefore, it is essential to investigate the molecular mechanisms underlying the pathogenesis of TSCC and to develop novel therapeutic approaches, in order to improve the diagnosis, treatment and prognosis of patients with TSCC.

MicroRNAs (miRNAs/miRs) are a large group of single-stranded, highly conserved, non-coding, short RNAs, 19–24 nucleotides in length, which are expressed in mammalian cells (9,10). miRNAs suppress the expression of their target genes through the action of the RNA-induced silencing complex, following binding of the miRNA molecule to the 3′ untranslated region (UTR) of its target mRNA; this results in the degradation of the target mRNA or the repression of mRNA translation (11). Current predictions suggest the existence of 800–1,000 miRNAs, which can regulate the expression of >30% of all human genes (12). Previous studies have demonstrated that miRNAs act as essential regulators of numerous biological processes, including cell growth, development, differentiation, apoptosis and endocrine homeostasis (13–15). The aberrant expression of cancer-associated miRNAs has been reported to participate in tumorigenesis and tumor development, via promoting the uncontrolled proliferation, enhancing the survival, inhibiting the differentiation and promoting the metastasis of cancer cells (16,17). Cancer-associated miRNAs have been suggested to function as oncogenes or tumor suppressors, depending on the type of tumor and their target genes (18). These findings suggested that miRNAs may be critical regulators of tumorigenesis and may have potential as novel therapeutic targets for the treatment of various types of human cancer.

Previous studies have reported that miR-509 is aberrantly expressed and may serve important roles in numerous types of human cancer (19–22). However, the expression levels and the biological roles of miR-509 in TSCC, as well as the molecular mechanisms underlying the effects of miR-509 on TSCC development and progression, have yet to be elucidated. Therefore, the present study aimed to assess the expression of miR-509 in TSCC tissues and cell lines, to explore the effects of miR-509 on TSCC cells, and to investigate the underlying molecular mechanisms that may be involved in the actions of miR-509.

Materials and methods

Clinical sample collection

Primary TSCC tissue samples and paired adjacent normal tissue samples were collected from 28 patients with TSCC (mean age, 57±9 years old; male, n=18; female, n=10; I–II stage, n=6; III–IV stage, n=22) undergoing surgery at the Department of Stomatology, Zaozhuang Municipal Hospital (Zaozhuang, China) between March 2012 and February 2014. None of the patients had received chemotherapy or radiotherapy prior to surgery. Tissue samples were trimmed, snap-frozen in liquid nitrogen and then stored at −80°C until RNA isolation. The present study was approved by the Ethics Committee of Zaozhuang Municipal Hospital, and written informed consent was obtained from all patients prior to enrollment in the present study.

Cell lines and culture conditions

Human Tca8113, SCC-15 and CAL-27 TSCC cells, and normal gingival epithelial cells (ATCC® PCS-200-014™) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). TSCC cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 mg/ml streptomycin. Normal gingival epithelial cells were cultured in minimum essential media (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 and 95% air.

Oligonucleotide transfection

miR-509 mimics and non-targeting negative control miRNA (miR-NC) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The miR-509 mimics sequence was 5′-UGAUUGGUACGUCUGUGGGUAG-3′ and the miR-NC sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. Small interfering (si)RNA targeting EGFR (si-EGFR) and the corresponding negative control siRNA (si-NC) were obtained from Shanghai Integrated Biotech Solutions Co., Ltd. (Ibsbio, Shanghai, China). The si-EGFR sequences were 5′-CCUUAGCAGUCUUAUCUAATT-3′ (forward) and 5′-AAGAGGACAAACCAGCCdTdT-3′ (reverse). The si-NC sequences were 5′-UUCUCCGAACGUGUCACGU-3′ (forward) and 5′-ACGUGACACGUUCGGAGAAdTdT-3′ (reverse). Tca8113 and CAL-27 cells, grown in DMEM containing 10% FBS as aforementioned, were seeded into 6-well plates at a density of 8×105 cells/well. miR-509 mimics (100 pmol) or miR-NC (100 pmol), and si-EGFR (100 pmol) or si-NC (100 pmol) were transfected into Tca8113 and CAL-27 cells using Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from tissue samples and cells (Tca8113, SCC-15, CAL-27 and normal gingival epithelial cells) using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. For miRNA expression, RT was performed using TaqMan MicroRNA Reverse Transcription kit, followed by qPCR using TaqMan PCR kit (both from Applied Biosystems; Thermo Fisher Scientific, Inc.). The temperature protocol for reverse transcription was as follows: 16°C for 30 min, 42°C for 30 min and 85°C for 5 min. The thermocycling conditions for qPCR were as follows: 50°C for 2 min, 95°C for 10 min, then 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 60 sec. For mRNA expression, cDNA was synthesized using PrimeScript RT Reagent kit and qPCR was performed using SYBR Premix Ex Taq II (both from Takara Biotechnology Co., Ltd., Dalian, China). The temperature protocol for reverse transcription was as follows: 37°C for 15 min and 85°C for 5 sec. The thermocycling conditions for qPCR were as follows: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec. U6 and GAPDH were used as housekeeping genes for miR-509 and EGFR mRNA expression, respectively. The primers were designed as follows: miR-509, 5′-TGCGGTACTGCAGACAGTGGCAA-3′ (forward) and 5′-CCAGTGCAGGGTCCGAGGT-3′ (reverse); U6, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ (forward) and 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (reverse); EGFR, 5′-GTGGCGGGACATAGTCAGCA-3′ (forward) and 5′-CCCATTGGGACAGCTTGGA-3′ (reverse); and GAPDH, 5′-CATGAGAAGTATGACAACAGCCT-3′ (forward) and 5′-AGTCCTTCCACGATACCAAAGT-3′ (reverse). Each sample was analyzed in triplicate and experiments were performed three times. Relative gene expression was quantified according to the comparative Cq method (23).

MTT assay

The effects of miR-509 overexpression and EGFR knockdown on TSCC cell proliferation were evaluated using an MTT assay (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Briefly, 3×103 cells/well were seeded into 96-well plates and transfected with miR-509 mimics, miR-NC, si-EGFR or si-NC, at 37°C in a humidified atmosphere containing 5% CO2 and 95% air for 0, 24, 48 and 72 h. At the indicated time points, an MTT assay was performed. MTT solution (20 µl; 5 mg/ml) was added to each well and cells were incubated at 37°C for 4 h. Subsequently, the culture medium was carefully removed and 150 µl dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) was added to each well. Following agitation for 10 min, the absorbance of each well at 490 nm was detected using a microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). Each sample was analyzed in triplicate and experiments were performed three times.

Invasion assay

Transwell chambers (8-µm pore size; Corning Incorporated, Corning, NY, USA) coated with Matrigel (BD Biosciences, San Jose, CA, USA) were used for the invasion assay. Following incubation at 37°C for 48 h, transfected cells were harvested using trypsinization and seeded into the upper chambers of the Transwell inserts at a density of 5×104 cells in 200 µl FBS-free DMEM, whereas the lower chambers were filled with 500 µl culture medium containing 20% FBS as a chemoattractant. Following incubation at 37°C for 48 h, non-invaded cells were removed using cotton swabs. Cells that had invaded into the lower membrane were fixed with 100% methanol at room temperature for 10 min, stained with 0.5% crystal violet at room temperature for 10 min, washed with PBS, air-dried and observed under an inverted microscope (Olympus Corporation, Tokyo, Japan). Photomicrographs were captured and invaded cells were counted by eye using an inverted microscope. Each sample was analyzed in triplicate.

miR-509 target prediction

The potential target genes of miR-509 were predicated using the online software miRanda (http://www.microrna.org) and TargetScan (http://www.targetscan.org).

Luciferase reporter assay

Luciferase reporter plasmids, pmirGLO-EGFR-3′UTR-wild type (Wt) or pmirGLO-EGFR-3′UTR-mutant (Mut), were synthesized by Shanghai GenePharma Co., Ltd. Human embryonic kidney (HEK)293T cells (ATCC) were seeded in 48-well plates at room temperature and grown overnight in DMEM with 10% FBS. Upon reaching 80–90% confluence, HEK293T cells were cotransfected with pmirGLO-EGFR-3′UTR-Wt (0.4 µg) or pmirGLO-EGFR-3′UTR-Mut (0.4 µg), and miR-509 mimics (10 pmol) or miR-NC (10 pmol) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature. A total of 48 h post-transfection, the cotransfected cells were harvested and luciferase activity was detected using a Dual-Luciferase Reporter assay system (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. Experiments were performed in triplicate.

Western blot analysis

Western blot analysis was performed using specific antibodies against EGFR (cat no. sc-71033; 1:1,000 dilution), Akt (cat no. sc-56878; 1:1,000 dilution), phosphorylated (p)-AKT (cat no. sc-514,032; 1:1,000 dilution), extracellular signal-regulated kinase (ERK; cat no. sc-514302; 1:1,000 dilution), p-ERK (cat no. sc-81492; 1:1,000 dilution) and GAPDH (cat no. sc-166574; 1:1,000 dilution) purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). GAPDH was used as the loading control. Total protein was extracted from transfected cells using Pierce cell lysis buffer (Pierce; Thermo Fisher Scientific, Inc.) and quantified using a bicinchoninic acid protein assay (Beyotime Institute of Biotechnology, Haimen, China). Equal amounts of extracted protein samples (20 µg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA), which were blocked overnight in 5% skim milk in TBS containing 0.05% Tween-20 (TBST) at room temperature. Subsequently, the membranes were incubated overnight at 4°C with the primary antibodies. Following washing with TBST, the membranes were incubated for 2 h at room temperature with a secondary antibody conjugated to horseradish peroxidase (1:5,000 dilution; cat no. sc-2005; Santa Cruz Biotechnology, Inc.), washed three times in TBST, and protein bands were visualized using enhanced chemiluminescence reagents (Pierce; Thermo Fisher Scientific, Inc.). Blots were semi-quantified by densitometry using Quantity One software version 4.62 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Statistical analysis

Data were expressed as the mean ± standard deviation of at least three repeated experiments. SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. The statistical significance of the differences between groups was assessed using Student's t-test or one-way analysis of variance followed by a Student-Newman-Keuls post hoc multiple comparisons test. Spearman's correlation analysis was used to evaluate the correlation between miR-509 and EGFR mRNA expression in TSCC tissues. P<0.05 was considered to indicate a statistically significant difference.

Results

miR-509 is downregulated in TSCC tissues and cell lines

The expression of miR-509 was detected in 28 TSCC tissues and paired adjacent normal tissue samples using RT-qPCR. As presented in Fig. 1A, miR-509 was significantly downregulated in TSCC tissues compared with in adjacent normal tissues (P<0.05). In addition, miR-509 expression was assessed in human TSCC cell lines and in normal gingival epithelial cells. As demonstrated in Fig. 1B, the expression levels of miR-509 were significantly decreased in the TSCC cell lines compared with in normal epithelial cells (P<0.05).

Figure 1. miR-509 expression in TSCC tissue samples and cell lines. (A) miR-509 expression was detected in 28 TSCC tissues and paired adjacent normal tissue samples. Data are presented as the mean ± standard deviation. *P<0.05 vs. adjacent normal tissues. (B) Expression levels of miR-509 in human Tca8113, SCC-15 and CAL-27 TSCC cell lines and normal gingival epithelial cells were detected using reverse transcription-quantitative polymerase chain reaction. Data are presented as the mean ± standard deviation. *P<0.05 vs. normal cells. miR, microRNA; TSCC, tongue squamous cell carcinoma.

miR-509 inhibits TSCC cell proliferation and invasion

To evaluate the effects of miR-509 on TSCC cell function, Tca8113 and CAL-27 cells, as they expressed relatively lower levels of miR-509, were transfected with miR-509 mimics or miR-NC. A total of 48 h post-transfection, RT-qPCR demonstrated that miR-509 was significantly upregulated in Tca8113 and CAL-27 cells transfected with miR-509 mimics compared with in miR-NC-transfected cells (P<0.05; Fig. 2A). An MTT assay revealed that the ectopic expression of miR-509 resulted in the inhibition of Tca8113 and CAL-27 cell proliferation compared with NC-transfected cells (P<0.05; Fig. 2B). In addition, an invasion assay revealed that the invasive capabilities of Tca8113 and CAL-27 cells were significantly suppressed following miR-509 overexpression (P<0.05; Fig. 2C). These findings suggested that miR-509 may act as a tumor suppressor in TSCC cells and inhibit their growth and metastasis.

Figure 2. miR-509 overexpression inhibits the proliferation and invasion of human Tca8113 and CAL-27 TSCC cells. (A) miR-509 expression was significantly upregulated in Tca8113 and CAL-27 cells transfected with miR-509 mimics compared with miR-NC-transfected cells. (B) Proliferation of Tca8113 and CAL-27 cells was inhibited following miR-509 overexpression. (C) Invasive capabilities of Tca8113 and CAL-27 cells transfected with miR-509 mimics were significantly suppressed (magnification, ×200). Data are presented as the mean ± standard deviation. *P<0.05 vs. miR-NC. miR, microRNA; NC, negative control; TSCC, tongue squamous cell carcinoma.

miR-509 directly targets EGFR in TSCC cells

To investigate the molecular mechanisms underlying the effects of miR-509 on TSCC cell proliferation and invasion, the putative targets of miR-509 were predicted using miRNA target prediction software. Among the potential target genes of miR-509, EGFR (Fig. 3A) has been reported to be overexpressed in TSCC cells, and has been correlated with TSCC development and progression (24,25). Therefore, a luciferase reporter assay was conducted; the results indicated that luciferase activity was significantly decreased in pmirGLO-EGFR-3′UTR-Wt-transfected cells that were cotransfected with miR-509 mimics (P<0.05; Fig. 3B). Conversely, no inhibition in luciferase activity was detected among cells transfected with the Mut EGFR 3′UTR sequence. In addition, RT-qPCR and western blot analysis demonstrated that transfection with the miR-509 mimic markedly decreased the mRNA and protein expression levels of EGFR in Tca8113 and CAL-27 cells (Fig. 3C and D). The present findings suggested that EGFR may be a direct target gene of miR-509 in TSCC cells.

Figure 3. EGFR is a direct target gene of miR-509 in TSCC cells. (A) Bioinformatics analysis predicted the miR-509-targeting sequence in the 3′UTR of EGFR mRNA. (B) A luciferase reporter assay was used to detect the interaction between miR-509 and the 3′UTR of EGFR mRNA. Human embryonic kidney 293T cells were transfected with pmirGLO-EGFR-3′UTR-Wt or pmirGLO-EGFR-3′UTR-Mut in the presence of miR-509 mimic or miR-NC. (C) mRNA and (D) protein expression levels of EGFR in human Tca8113 and CAL-27 TSCC cells transfected with miR-509 mimics or miR-NC. Data are presented as the mean ± standard deviation. *P<0.05 vs. miR-NC. (E) Western blot analysis confirmed that EGFR protein expression was downregulated in Tca8113 and CAL-27 cells following transfection with si-EGFR. (F) Proliferation of Tca8113 and CAL-27 cells was inhibited following EGFR knockdown. (G) Invasive capabilities of Tca8113 and CAL-27 cells transfected with si-EGFR were significantly suppressed (magnification, ×200). Data are presented as the mean ± standard deviation. *P<0.05 vs. si-NC. EGFR, epidermal growth factor receptor; miR, microRNA; Mut, mutant; NC, negative control; si, small interfering; TSCC, tongue squamous cell carcinoma; UTR, untranslated region; Wt, wild-type.

To investigate the roles of EGFR on TSCC cell biological activity, si-EGFR was used to knock down the expression of EGFR in Tca8113 and CAL-27 cells. Successful silencing was confirmed by western blot analysis (P<0.05; Fig. 3E). An MTT assay demonstrated that EGFR knockdown significantly inhibited the proliferation of Tca8113 and CAL-27 cells compared with si-NC-transfected cells (P<0.05; Fig. 3F). Furthermore, a Transwell invasion assay revealed that the downregulation of EGFR expression significantly suppressed the invasive capabilities of Tca8113 and CAL-27 cells (P<0.05; Fig. 3G). These findings suggested that miR-509 may regulate the progression of TSCC, through the direct regulation of EGFR expression.

EGFR mRNA expression is negatively correlated with miR-509 expression in TSCC tissues

In the present study, EGFR was identified as a direct target gene of miR-509 in TSCC cells; therefore, the expression of EGFR was assessed in TSCC tissues and in paired adjacent normal tissues, and the correlation between EGFR and miR-509 expression was investigated. As presented in Fig. 4A, EGFR mRNA expression was significantly upregulated in TSCC tissues compared with in adjacent normal tissue samples (P<0.05). Furthermore, an inverse correlation was detected between EGFR mRNA and miR-509 expression in TSCC tissues (r=−0.5109, P=0.0055; Fig. 4B). The present findings suggested that miR-509 may be involved in the development and progression of TSCC, through the negative regulation of EGFR.

Figure 4. EGFR mRNA and miR-509 expression levels are negatively correlated in TSCC tissues. (A) EGFR mRNA expression levels were detected in 28 TSCC tissues and paired adjacent normal tissue samples. Data are presented as the mean ± standard deviation. *P<0.05 vs. adjacent normal tissues. (B) Spearman's correlation analysis revealed an inverse correlation between miR-509 and EGFR mRNA expression in TSCC tissues. EGFR, epidermal growth factor receptor; miR, microRNA; TSCC, tongue squamous cell carcinoma.

miR-509 suppresses EGFR-associated signaling in TSCC cells

Since EGFR was identified as a direct target gene of miR-509, the effects of miR-509 upregulation were examined on the signaling pathways downstream of EGFR. As demonstrated in Fig. 5, miR-509 overexpression downregulated the protein expression levels of p-Akt (P<0.05) and p-ERK (P<0.05) in Tca8113 and CAL-27 cells, whereas it exerted no significant effect on the expression of Akt and ERK protein expression. The present results suggested that miR-509 may inhibit EGFR-associated signaling pathways in TSCC cells.

Figure 5. miR-509 overexpression suppresses the expression of p-Akt and p-ERK in Tca8113 and CAL-27 TSCC cells. The protein expression levels of Akt and ERK remained unaltered following transfection with miR-509 mimics or miR-NC. *P<0.05 vs. NC. ERK, extracellular signal-regulated kinase; miR, microRNA; NC, negative control; p-, phosphorylated; TSCC, tongue squamous cell carcinoma.

Discussion

miRNA-based therapeutic approaches may have potential as effective anticancer treatment strategies, through the regulation of target genes that are implicated in numerous physiological and pathological processes (26,27). Therefore, it is essential to investigate the expression, roles and underlying molecular mechanisms of cancer-associated miRNAs in humans, and develop novel therapeutic targets for miRNA-based cancer treatment strategies. In the present study, miR-509 was revealed to be significantly downregulated in TSCC tissues and cell lines compared with in normal tissues and normal epithelial cells, respectively. Functional assays demonstrated that overexpression of miR-509 inhibited the proliferation and invasion of TSCC cells in vitro. Furthermore, EGFR was identified as a direct target gene of miR-509 in TSCC cells. miR-509 overexpression also resulted in the downregulation of p-AKT and p-ERK expression in TSCC cells, thus suggesting that EGFR-associated signaling were inhibited. The present findings indicated that miR-509 may exert tumor-suppressive roles in TSCC cells, through the regulation of EGFR-mediated signaling pathways.

Previous studies have identified miR-509 as a critical regulator of tumorigenesis and tumor development, via acting as a tumor suppressor gene in numerous types of human cancer: Zhai et al (28) reported that miR-509 was downregulated in renal carcinoma tissue samples, whereas the restoration of miR-509 expression attenuated the proliferation and migration, and induced the apoptosis of renal carcinoma cells. Su et al (19) also demonstrated that miR-509 expression was decreased in renal carcinoma cells, whereas its upregulation resulted in the significant suppression of cancer cell proliferation and migration. A study by Chen et al (20) revealed that miR-509 expression levels were decreased in chemoresistant epithelial ovarian cancer tissues. Conversely, the overexpression of miR-509 suppressed the proliferation and migration of ovarian cancer epithelial cells, disrupted multi-cellular spheroids and improved their sensitivity to cisplatin-induced apoptosis (20,29). In addition, miR-509 expression has been revealed to be downregulated in gastric cancer tissues, and low miR-509 expression has been associated with decreased overall survival of patients with gastric cancer; conversely, the restoration of miR-509 expression suppressed gastric cancer cell motility (21). Zhang et al (22) reported that miR-509 served tumor-suppressive roles in triple-negative breast cancer cell proliferation, invasion and apoptosis. These studies suggested that miR-509 may have potential as a novel therapeutic target for the development of cancer treatments.

The molecular mechanisms underlying the anti-proliferative and anti-invasive effects of miR-509 in TSCC cells were investigated in the present study. Several targets of miR-509 have been identified, including mitogen-activated protein kinase kinase kinase 8 (19), X-linked inhibitor of apoptosis protein (20,21), tumor necrosis factor-α (22), cyclin-dependent kinase 2 (30), Ras-related C3 botulinum toxin substrate 1 (30), and phosphatidylinositol-4-phosphate 3-kinase C2 domain-containing α polypeptide (30). In the present study, bioinformatics analysis identified numerous candidate target genes for miR-509. Among them, EGFR contained a putative binding site for miR-509 in its 3′UTR, and has been reported to be upregulated in TSCC cells, where it contributes to the development and progression of TSCC (24,25). Therefore, the present study investigated whether the tumor-suppressive roles of miR-509 in TSCC cells may be attributed to the negative regulation of EGFR. Luciferase reporter assays demonstrated that miR-509 directly targeted the 3′UTR of EGFR. In addition, the mRNA and protein expression levels of EGFR were revealed to be downregulated in TSCC cells following transfection with miR-509 mimics, whereas EGFR knockdown suppressed the proliferation and invasion of TSCC cells, similar to miR-509 overexpression. Furthermore, an inverse correlation was revealed between miR-509 and EGFR mRNA expression in TSCC tissues, whereas the upregulation of miR-509 expression was revealed to inhibit EGFR-associated signaling pathways in TSCC cells. Taken together, these findings suggested that the downregulation of EGFR expression and the inhibition of its downstream signaling pathways may be important mechanisms implicated in the miR-509 induced suppression of TSCC development and progression.

EGFR is a cell-surface receptor for members of the EGF family and transduces critical growth factor signals from the extracellular to intracellular environment (31). EGFR has been reported to be activated by various ligands, including EGF, transforming growth factor-α, amphiregulin, heparin-binding EGF, betacellulin and epiregulin (32). In addition, EGFR has been revealed to be abnormally upregulated in numerous tumor types, including colorectal (33), gastric (34) and bladder cancer (35), and osteosarcoma (36). Increasing evidence has indicated that the upregulation of EGFR is strongly correlated with tumor development and progression, and poor disease prognosis (37–40). In TSCC, Ulanovski et al (24) reported that EGFR was highly expressed in tumor specimens and its expression was obviously correlated with tumor differentiation. Nakata et al (41) revealed that the expression levels of EGFR were significantly associated with reduced disease-free survival and overall survival of patients with TSCC. Previous functional assays also indicated an oncogenic role for EGFR during TSCC cell growth, metastasis and apoptosis (42,43). These findings suggested that targeting EGFR may have potential as a novel and efficient therapeutic strategy for the treatment of patients with TSCC.

In conclusion, the present study was, to the best of our knowledge, the first to demonstrate that miR-509 acted as a tumor suppressor in TSCC, via negatively regulating EGFR and inhibiting its downstream signaling pathways. Further studies are required to fully elucidate the roles of miR-509 and EGFR in TSCC and reveal novel strategies for the treatment of this malignancy.

References