MicroRNA‑206 exerts anti‑oncogenic functions in esophageal squamous cell carcinoma by suppressing the c‑Met/AKT/mTOR pathway.

Increasing evidence suggests that the dysregulation of microRNAs (miRNAs) has an important role in the progression of human cancer, including ESCC. However, the exact functions and mechanisms of miRNAs in ESCC remain largely unclear. The aim of the present study was to investigate the expression and biological functions of miRNAs in ESCC and reveal the underlying molecular mechanisms. miRNA microarray and reverse transcription‑quantitative polymerase chain reaction analyses were performed, which identified and confirmed that miR‑206 was significantly downregulated in ESCC tissues and cell lines. Its low expression was associated with lymph node metastasis, advanced TNM stage and N classification, as well as poorer overall survival in patients with ESCC. CCK‑8 and flow cytometry assays demonstrated that ectopic miR‑206 expression inhibited ESCC cell proliferation and induced cell apoptosis. In addition, MET proto‑oncogene, receptor tyrosine kinase (c‑Met), a well‑known oncogene, was a direct target of miR‑206. An inverse correlation between the levels of miR‑206 and c‑Met mRNA in ESCC tissue samples was confirmed. Notably, c‑Met overexpression inhibited the effects of miR‑206 on the proliferation and apoptosis of ESCC cells. Additionally, it was confirmed that the tumor‑suppressive functions of miR‑206 may have contributed to the inactivation of the c‑Met/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway. In conclusion, the findings of the present study suggested that miR‑206 exerts its anti‑cancer functions via the c‑Met/AKT/mTOR signaling pathway, providing a novel candidate prognostic factor and a potential therapeutic target in ESCC.

Introduction

Esophageal cancer remains one of the leading causes of cancer mortality worldwide, with approximately 477,900 new cases of esophageal cancer diagnosed in 2012 (1). One of the major types of esophageal cancer is esophageal squamous cell carcinoma (ESCC), which accounts for ~90% of esophageal cancers (2,3). Although a number of improvements have been achieved in diagnosis and treatment, ESCC remains a devastating malignancy (4). Patients with ESCC have high terminal metastasis and poor prognosis (5). Therefore, further understanding of the underlying biological mechanisms of ESSC and the identification of feasible therapeutic targets for ESCC is urgently required.

MicroRNAs (miRNAs/miRs) are a conserved group of small non-coding RNAs comprising of 18–22 nucleotides that regulate gene expression at the post-transcriptional level (6). miRNAs have emerged as effective regulators of cell proliferation, apoptosis and differentiation (7,8). In ESCC, several miRNAs have been identified as effective tumor biomarkers for cancer diagnosis, and have oncogenic or tumor suppressor functions (9–12). For example, Sun et al (13) reported that miR-367 expression is frequently downregulated in ESCC, and its downregulation is significantly correlated to advanced disease progression and poor patient prognosis. miR-214 was also demonstrated to be downregulated in ESCC and further studies have revealed that miR-214 markedly represses ESCC cell proliferation by regulating cell division cycle 25B (14). Therefore, examining the roles of other miRNAs in ESCC may provide novel avenues for the early diagnosis, therapy and prognosis of ESCC.

In the present study, miRNA microarray analysis to investigate miRNA expression in human ESCC tissue. miR-206 was identified as a differentially expressed miRNA and selected for further study in ESCC tissues and cells as miR-206 has been demonstrated to be a well-known tumor suppressor (15,16). Subsequently, the mechanisms underlying the effects of miR-206 on ESCC cell proliferation and apoptosis were investigated, and the collective signaling pathways enriched by the predicted targets of miR-206 in ESCC carcinogenesis were analyzed. The findings of the present study suggested that miR-206 may be a potential target for the treatment of ESCC.

Materials and methods

Clinical specimens

ESCC and paired normal esophageal tissues (>5 cm from the tumor margin) were obtained from 52 patients (median age 63.5 years, range 48–76 years, female:male=9:17) who underwent esophagus resection between January 2014 and May 2016 at the Department of Thoracic Surgery, the Second Affiliated Hospital of Zhengzhou University (Zhengzhou, China). Patient characteristics are presented in Table I. The ESCC patients were recruited according to the following inclusion and exclusion criteria: First, all ESCC patients were newly diagnosed and confirmed by histopathological examination in accordance with the 7th edition of the TNM-UICC/AJCC classification (17); second, all included patients underwent a total or subtotal esophagectomy with resection of at least 12 regional lymph nodes; third, all enrolled patients were diagnosed and had not received any other treatment before esophagectomy; fourth, follow-up data could be obtained from all eligible patients. Patients with acute or chronic infection, autoimmune, hematological or liver disease or other malignancies and those without clinical characteristics or survival data were excluded in the present study. Following excision, tissue specimens were immediately frozen in liquid nitrogen for subsequent analysis. Informed consent was obtained, and the present study was approved by the Ethics Committee of the Second Affiliated Hospital of Zhengzhou University (Zhengzhou, China; approval no. 2016–008).

Table I.

Correlation between miR-206 expression and the clinicopathological features of patients with esophageal squamous cell carcinoma.

		miR-206
Clinical variables	Total n=52	High expression	Low expression	P-value
Gender				0.7165
Male	34	15	19
Female	18	7	11
Age (years)				0.8612
≥60	30	13	17
<60	22	9	13
Tumor location				0.5386
Upper or middle 1/3	40	16	24
Lower 1/3	12	6	6
Lymph node metastasis				0.0015[b]
Negative	16	12	4
Positive	36	10	26
TNM stage				0.0209[a]
I–II	19	12	7
III–IV	33	10	23
Differentiation				0.4043
Well and moderate	37	17	20
Poor	15	5	10
Distant metastasis				0.3807
M0	42	19	23
M1	10	3	7

P<0.05

P<0.01. miR-206, microRNA-206.

Microarray analysis

Total RNA was extracted from ESCC tissues and adjacent normal tissues using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and Cy3- or Cy5-labeled cDNAs were hybridized on the miRCURY™ LNA Array kit (version 16.0; Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's instructions. Following washing with PBS, the slides were scanned using an Axon GenePix 4000 B microarray scanner (Molecular Devices, LLC, Sunnyvale, CA, USA). Scanned images were subsequently imported into the GenePix Pro 6.0 program (Molecular Devices, LLC) for grid alignment and data extraction. Replicated miRNAs were averaged, and miRNAs with intensities of ≥50 in all samples were used to calculate a normalization factor. Expressed data were normalized by median normalization. Following this, the miRNAs that were significantly differentially expressed were identified by Volcano Plot filtering with fold change values >2 and P<0.05 as the screening conditions using the R software package (version 3.1.3, 2015; R Core Team, Vienna, Austria). Finally, the expression data were subjected to hierarchical clustering and subsequently depicted in a heat map format using GeneSpring GX (version 7.3; Agilent Technologies, Inc., Santa Clara, CA, USA).

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

miRNA was isolated from tissue samples and cell lines using the mirVANA RNA isolation kit (Ambion; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was reverse transcribed into complementary DNA (cDNA) by using a miScript reverse transcription kit (Qiagen, Inc.) in a volume of 20 µl containing 10 µl RNA, 4 µl miScript RT Buffer, 1 µl miScript Reverse Transcriptase Mix and 6 µl RNase-free H2O. The reaction was incubated for 1 h at 37 °C. miRNA PCR was performed on an Applied Biosystems Prism 7900HT Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Relative expression was determined by normalization to U6. The primers for RT-qPCR analysis were as follows: miR-206 forward, 5′-TGGAATGTAAGGAAGTG-3′ and reverse, 5′-CAGTGCGTGTCGTGGAGT-3′; U6 forward, 5′-TGCGGGTGCTCGCTTCGCAGC-3′ and reverse, 5′-CCAGTGCAGGGTCCGAGGT-3′. The amplification protocol was set as follows: Initial denaturation at 95°C for 5 min, followed by 50 cycles of denaturation at 94°C for 15 sec, annealing at 55°C for 30 sec and extension at 70°C for 30 sec.

For mRNA reverse transcription, cDNA was synthesized using Primescript RT Reagent (Takara Bio, Inc.). The RT-qPCR reaction system (30 µl) contained 5 µl cDNA, 15 µl 2X qPCR mix, 1 µl upstream primer, 1 µl downstream primer and 8 µl double distilled H2O. PCR was performed with an Applied Biosystems Prism 7900HT Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the protocol was: 95°C for 15 min, followed by 40 cycles of 94°C for 15 sec, 55°C for 30 sec and 70°C for 30 sec with a final extension step of 72°C for 5 min using. Relative quantification was determined by normalization to U6 or GAPDH. The primer sequences were as follows: c-Met, forward 5′-CCCCACCCTTTGTTCAG-3′ and reverse 5′-TCAGCCTTGTCCCTCCT-3′; GAPDH forward, 5′-GAAGATGGTGATGGGATTTC-3′, and reverse, 5′-GAAGGTGAAGGTCGGAGT-3′; RT-qPCR assays were performed in triplicate and expression alterations were calculated using the 2−ΔΔCq method (18).

Cell lines and cultures

ESCC cell lines Ec9706, Eca109 and KYSE410, as well as the normal esophageal cell line Het-1A, were obtained from the American Type Culture Collection (Manassas, VA, USA). All the cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere of 95% air and 5% CO2.

Cell transfection

miR-206 mimics, miR-206 inhibitor, and their negative controls (NCs) were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). The coding domain sequences of c-Met mRNA were amplified by PCR, and inserted into a pcDNA 3.0 vector (Invitrogen; Thermo Fisher Scientific, Inc.) to enhance its expression, and was termed pcDNA-c-Met. Eca109 and KYSE410 cells (5.0×105/well) were seeded and cultured to 60–80% confluence in six-well plates. According to the manufacturer's protocol, they were transiently transfected with the 20 nM interference oligonucleotide which included negative control (inhibitor NC: 5′-CAGUACUUUUGUGUAGUACAA-3′; mimics NC: 5′-UUCUCCGAACGUGUCACGUTT-3′), miR-206 inhibitor (5′-CCACACACUUCCUUACAUUCCA-3′) or miR-206 mimics (5′-UGGAAUGUAAGGAAGUGUGUGG-3′) using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Following 6 h transfection at 37°C, the medium was replaced with RPMI1640 media containing 10% FBS and the cells were harvested for further experiments after 48 h.

Cell proliferation

A Cell Counting Kit-8 (CCK-8) assay was performed to assess the proliferation of Eca109 and KYSE410 cells following transfection in 96-well plates at a density of 5×103 cells/well. After 24, 48 and 72 h of incubation, 10 µl CCK-8 was added to each well. After incubation for 4 h at 37°C, the absorbance rates were measured at a wavelength of 450 nm using a microplate reader (Infinite M200; Tecan Group, Ltd., Mannedorf, Switzerland). All experiments were performed in triplicate.

Cell apoptosis

Cell apoptosis was determined using an Annexin V-FITC Apoptosis Detection kit (cat no. ab14085; Abcam, Cambridge, UK) according to the manufacturer's protocol. Briefly, Eca109 and KYSE410 cells were seeded in 6-well plates at a density of 1.0×106 cell/well, and subjected to the various treatments as described above. At the end of the exposure, the cells were harvested and washed twice with PBS, and stained with 5 µl of FITC Annexin V (Abcam, Cambridge, UK), and 5 µl of propidium iodide (PI; Abcam, Cambridge, UK) in the dark for 15 min at room temperature. Following this, cell apoptosis was analyzed on Becton Dickinson FACScan flow cytometer (Beckman Coulter, Inc., Brea, CA, USA). Data was analyzed with the ModFit LT software package version 4.0 (Verity Software House, Inc., Topsham, ME, USA).

Target genes prediction of miR-206

The target genes of miR-206 were predicted by bioinformatics analysis. The analysis was performed by TargetScan 5.1 (targetscan.org) (19), miRanda (2010 release; microRNA.org) (20) and PicTar 5 (pictar.mdc-berlin.de) (21). Results of the forecast targets were intersected by miRWalk V2.0 (www.ma.uni-heidelberg.de/apps/zmf/mirwalk) website tools (22).

Dual-luciferase reporter assays

The predicted and mutated sequences targeting the 3′untranslated region (UTR) of c-Met were synthesized and ordered from Shanghai GenePharma Co., Ltd. 293T cells (1×104 cells/well) in 24-well plates were co-transfected with miR-206 mimics (50 nM), mimics NC (50 nM), miR-206 inhibitor (50 nM), inhibitor NC (50 nM) and luciferase reporter plasmids (50 ng) (pmiRGLO-c-Met-3′UTR Wt or pmiRGLO-c-Met-3′UTR Mut) using Lipofectamine® 2000, according to the manufacturer's recommendations. The relative luciferase activities were determined using a Dual-Luciferase Reporter Assay system (Promega Corporation, Madison, WI, USA) at 48 h post-transfection. All experiments were performed in triplicate. Renilla luciferase activity was normalized to firefly luciferase activity.

Western blotting

Following transfection, total protein was extracted from cells using radioimmunoprecipitation assay (RIPA) lysis buffer containing 1× protease inhibitory cocktail and phosphatase inhibitors (RIPA Lysis Buffer system, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Protein concentration was determined using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology, Haimen, China). Proteins (25 µg/lane) were analyzed by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (GE Healthcare, Chicago, IL, USA) by electroblotting. The membranes were blocked with 5% bovine serum albumin (BSA, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 1 h at room temperature. Subsequently, the membranes were incubated with primary antibodies at 4°C overnight. Primary antibodies against c-Met (cat no. 8198; 1:1,000; Santa Cruz Biotechnology, Inc.), phosphorylated (p)-c-Met (cat no. ab5662; 1:1,000; Abcam; Cambridge, UK), p-mechanistic target of rapamycin (p-mTOR; cat no. 5536; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), mTOR (cat no. 2983; 1:1,000; Cell Signaling Technology, Inc.), p-AKT (cat no 4060; 1:1,000; Cell Signaling Technology, Inc.), AKT (cat no. 4691; 1:1,000; Cell Signaling Technology, Inc.) and β-actin (cat no. A1987; 1:2,000; Sigma-Aldrich; Merck KGaA). Thereafter, membranes were incubated with secondary antibodies (cat no. 8885; 1:10,000; Cell Signaling Technology, Inc.). Immunoreactivity was visualized using an Enhanced Chemiluminescence Western Blotting kit (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturer's protocols. The intensity of the bands was analyzed by ImageJ software (version 1.46; National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

Statistical analysis was performed with SPSS (version 18.0; SPSS, Inc., Chicago, IL, USA). Data were presented as the mean ± standard deviation. Student's t-test or one-way analysis of variance followed by Tukey's post-hoc test was used to analyze the differences among/between sample groups. Kaplan-Meier analysis was performed to calculate the overall survival (OS) rates, and a log-rank test was conducted to compare the survival distributions between two groups. Multivariate analysis for overall survival (OS) was performed using the Cox proportional hazard model, with factors identified to be statistically significant from univariate analysis. Correlations between miR-206 and c-Met expression levels in databases were assessed by Spearman's correlation. P≤0.05 was considered to indicate a statistically significant difference.

Results

miR-206 expression is downregulated in ESCC tissues and cell lines, and correlated with clinicopathologic features

To determine the potential involvement of miRNAs in ESCC, miRNA microarray profiling was performed in ESCC tissues and matched normal tumor-adjacent tissues. The data revealed that compared with the normal group, 35 miRNAs were upregulated and 22 miRNAs were downregulated in the ESCC group (Fig. 1A). Among these differentially expressed miRNAs, miR-206 was one of the most downregulated miRNAs, and several studies have previously demonstrated that miR-206 suppresses cell growth and invasion in various types of cancers (23–25). However, the function and mechanism of miR-206 in ESCC has not been characterized. Thus, miR-206 was selected for further analysis.

Figure 1.

miR-206 is downregulated in ESCC tissues and correlated with clinicopathologic features. (A) Heatmap of normalized expression levels of miRNAs in ESCC tissues and matched tumor-adjacent tissues. Green indicates low expression levels; red indicates high expression levels. (B) miR-206 expression was detected by RT-qPCR in ESCC tissues (n=52) and normal matched tumor-adjacent tissues (n=52). P<0.01 vs. normal group. (C) miR-206 expression was detected by RT-qPCR in three ESCC cell lines (Ec9706, Eca109 and KYSE410) and a normal esophageal cell line (Het-1A). Data are represented the mean ± standard deviation of three independent experiments. **P< 0.01 vs. Het-1A. (D) Overall survival of patients with high or low miR-206 expression. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; miR/miRNA, microRNA; ESCC, esophageal squamous cell carcinoma.

Next, the expression levels of miR-206 in 52 paired ESCC tissues and matched tumor-adjacent tissues were validated by RT-qPCR. Consistent with the array data, miR-206 expression was significantly lower in ESCC tissues compared with that in the matched tumor-adjacent tissues (Fig. 1B). The expression of miR-206 was also examined in a series of ESCC cell lines (Ec9706, Eca109 and KYSE410). The results revealed that that the expression of miR-206 was markedly downregulated in these cell lines when compared with the normal Het-1A cell line, particularly in Eca109 and KYSE410 cells (Fig. 1C). These data suggested that miR-206 may function as a tumor suppressor in the initiation and progression of ESCC.

In order to evaluate the clinical importance of miR-206 in ESCC, the association between miR-206 expression levels and the clinicopathological parameters of ESCC patients was investigated. A total of 52 ESCC patients were divided into two groups according to the median level of miR-206 (cut off value for miR-206 was 2.25). As presented in Table I, low expression of miR-206 was inversely associated with lymph node metastasis and TNM stage. However, there were no significant correlations between miR-206 expression levels with gender, age, tumor location, differentiation or distant metastasis in ESCC. In addition, it was demonstrated that patients with low miR-206 expression had shorter OS compared with patients with high miR-206 expression (Fig. 1D). These results indicated that miR-206 downregulation may be associated with the development of ESCC.

Overexpression of miR-206 inhibits cell proliferation and induces cell apoptosis

Considering that miR-206 was downregulated in ESCC tissues, it was hypothesized that miR-206 may function as a tumor suppressor in ESCC. To test this hypothesis, overexpression of miR-206 was established in Eca109 and KYSE410 cells, which had the lowest expression of miR-206 in the three ESCC cell lines. The efficiency of miR-206 mimic transfection in Eca109 and KYSE410 cells was confirmed through RT-qPCR (Fig. 2A). The results of the CCK-8 assay demonstrated that miR-206 overexpression significantly inhibited cell proliferation in Eca109 (Fig. 2B) and KYSE410 cells (Fig. 2C), compared with the mimic NC groups. To further understand the mechanisms by which cell proliferation was affected, flow cytometry was performed to analyze cell apoptosis. As presented in Fig. 2D, cell apoptosis was markedly promoted in the miR-206 mimics group compared with mimics NC group. These results implied that miR-206 may have inhibited ESCC cell proliferation by promoting apoptosis.

Figure 2.

Overexpression of miR-206 suppresses cell proliferation and induces cell apoptosis. Eca109 and KYSE410 cells were transfected with miR-206 mimics and mimics NC for the indicated durations and cells were subsequently harvested for further experimentation. (A) The transfection efficiency of miR-206 mimics was evaluated by reverse transcription-quantitative polymerase chain reaction analysis. (B and C) Cell proliferation was measured by a Cell Counting Kit-8 assay in Eca109 and KYSE410 cells. (D) Cell apoptosis was determined by flow cytometry. Data are represented the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 vs. mimics NC group, ##P<0.01 vs. inhibitor NC. miR, microRNA; NC, negative control; FITC, fluorescein isothiocyanate; OD, optical density; PI, propidium iodide.

c-Met is a direct target of miR-206

To further elucidate the underlying molecular mechanisms involved in the anti-oncogenic role of miR-206 in ESCC cells, target genes of miR-206 were searched for using TargetScan 5.1, miRanda (19) and PicTar 5 (26). The predicted results indicated that miR-206 directly targeted the c-Met gene and that the target sequences were highly conserved among different species (Fig. 3A and B). To confirm whether c-Met was a direct target of miR-206, a dual-luciferase reporter system containing the 3′-UTR of c-Met along with the putative miR-206 binding sites was constructed.

Figure 3.

c-Met is a direct target of miR-206. (A) The evolutionarily conserved sites of c-Met 3′-UTR were targeted by miR-206 among several different species. The schematic was obtained from TargetScan (B) The putative binding site of miR-206 and c-Met. (C) Luciferase assay of 293T cells co-transfected with firefly luciferase constructs containing the c-Met wild-type or mutated 3′-UTRs and miR-206 mimics, inhibitor or NCs, as indicated (n=3). Data are represented as the mean ± standard deviation of three independent experiments. **P<0.01 vs. mimics NC, ##P<0.01 vs. inhibitor NC. (D) c-Met protein expression, following transfection with miR-206 mimics or miR-206 inhibitor, was measured by western blot analysis and the bands were analyzed using ImageJ software, normalized to β-actin band density. **P<0.01 vs. mimics NC, ##P<0.01 vs. inhibitor NC. (E) c-Met mRNA expression was determined in ESCC tissues and matched normal adjacent tissues. P<0.01 vs. normal group. (F) c-Met mRNA expression was determined in ESCC cell lines. Data represents the mean ± standard deviation of three independent experiments. **P<0.01 vs. Het-1A. (G) Correlations between miR-206 and c-Met expression levels in ESCC tissues were assessed by Spearman's correlation (R2=0.7922; P<0.001). miR, microRNA; ESCC, esophageal squamous cell carcinoma; c-Met, MET proto-oncogene, receptor tyrosine kinase; 3′UTR, 3′ untranslated region; NC, negative control; wt, wild type; mut, mutated.

The results of the luciferase reporter gene assay demonstrated that the luciferase activity was remarkably decreased following co-transfection with the luciferase reporter plasmid harboring the c-Met 3′UTR wild-type (WT) and miR-206 mimics. By contrast, luciferase activity was notably increased following co-transfection with c-Met 3′UTR wild-type (WT) plasmid and miR-206 inhibitor. However, these effects were abrogated when the c-Met sequence was mutated (Fig. 3C). To further identify the correlation between miR-206 expression levels and c-Met, c-Met protein expression was determined in Eca109 and KYSE410 cells by western blotting. The expression of c-Met at the protein level was significantly downregulated following overexpression of miR-206, whereas it was upregulated following knockdown of miR-206 in Eca109 and KYSE410 ESCC cells (Fig. 3D). In addition, the results revealed that c-Met expression at the mRNA level was significantly upregulated in ESCC tissues compared with that in matched normal adjacent tissues (Fig. 3E), as well as increased in the three ESCC cell lines, compared with the normal esophageal cell line (Fig. 3F). Furthermore, miR-206 expression was inversely correlated with c-Met expression in ESCC tissues (R2=0.7922; P<0.001; Fig. 3G). These data suggested that miR-206 suppressed the expression of c-Met in ESCC.

Overexpression of c-Met inhibits the tumor suppressive role of miR-206 in ESCC cells

Given that c-Met is a target of miR-206, and c-Met has been widely documented to regulate proliferation and migration during the development of human cancers (27), whether miR-206 mediated the inhibitory effects on ESCC via c-Met was examined. First, the effects of c-Met overexpression in miR-206 mimic transfected cells were investigated. Western blotting results demonstrated that pcDNA-c-Met vector transfection significantly increased c-Met expression in miR-206 mimic transfected Eca109 and KYSE410 cells (Fig. 4A). Subsequently, cell proliferation was assessed with a CCK-8 assay. As presented in Fig. 4B and C, overexpression of c-Met in Eca109 and KYSE410 cells partly suppressed the inhibitory effects of miR-206 on cell proliferation. In addition, it was demonstrated that overexpression of c-Met abrogated the promotive effects of miR-206 on cell apoptosis in Eca109 and KYSE410 cells (Fig. 4D and E). These data suggested that miR-206 exerted its tumor suppressive effects by modulating c-Met expression.

Figure 4.

c-Met restoration prevents the inhibitory effects of miR-206 overexpression on squamous cell carcinoma cells. Eca109 and KYSE410 cells were co-transfected with miR-206 mimics and pcDNA-c-Met or their relative controls for the indicated times prior to harvesting of cells for further experiments. (A) The protein expression of c-Met was determined by western blot analysis. (B) Cell proliferation was measured with a Cell Counting Kit-8 assay in Eca109 and (C) KYSE410 cells. (D) Cell apoptosis was determined by flow cytometry in Eca109 and KYSE410, and (E) apoptotic percentage was calculated. Data represent the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 vs. mimics NC group, #P<0.05, ##P<0.01 vs. miR-206 mimics group. miR, microRNA; c-Met, MET proto-oncogene, receptor tyrosine kinase; NC, negative control; FITC, fluorescein isothiocyanate; OD, optical density; PI, propidium iodide.

Overexpression of miR-206 blocked the activation of c-Met/AKT/mTOR pathway

To investigate the molecular mechanism underlying the inhibition of cell proliferation by miR-206, The expression of c-Met, p-c-Met, AKT, p-AKT, mTOR and p-mTOR was determined by western blot analysis (Fig. 5A). The results revealed that overexpression of miR-206 inhibited the p-c-Met, p-AKT and p-mTOR expression, whereas the expressions of Met, AKT and mTOR were not noticeably altered in Eca109 or KYSE410 cells (Fig. 5B). These data suggested that miR-206 regulated the c-Met/AKT/mTOR pathway, which was at least partially responsible for the effects of miR-206 on cell proliferation and apoptosis in ESCC cells.

Figure 5.

Overexpression of miR-206 inhibits the activation of the AKT/mTOR pathway. Eca109 and KYSE410 cells were transfected with miR-206 mimics or mimics NC for 48 h and subjected to western blot analysis. (A) c-Met, p-c-Met, AKT, p-AKT, p-mTOR and mTOR were detected by western blotting and (B) bands were quantitatively analyzed using ImageJ software, normalized to β-actin density. Data represent the mean ± standard deviation of three independent experiments. **P< 0.01 vs. mimics NC. miR, microRNA; AKT, protein kinase B; mTOR, mechanistic target of rapamycin; -Met, MET proto-oncogene, receptor tyrosine kinase; NC, negative control; p, phosphorylated.

Discussion

In the present study, miR-206 was significantly downregulated in ESCC and its expression was correlated with poor prognosis. Overexpression of miR-206 inhibited ESCC cell proliferation and induced ESCC cell apoptosis. In addition, miR-206 upregulation in ESCC cells inactivated the AKT/mTOR signaling pathway by targeting c-Met. The data from the present study indicated that miR-206 may be a novel biomarker and therapeutic target in ESCC.

Increasing evidence has revealed that miRNAs have pivotal function in cancer development and progression, including ESCC (28–31). In the present study, differentially expressed miRNAs were profiled in ESCC tissues, and it was demonstrated that miR-206 was one of most downregulated miRNAs. miR-206 was selected for further analysis, as miR-206 has important involvement in various types of cancers. For example, the expression levels of miR-206 are markedly decreased in breast cancer tissues, and low miR-206 expression is associated with advanced disease progression and poor prognosis (32). Pan et al (24) demonstrated that miR-206 suppresses medulloblastoma cell viability and invasion, at least partially via the targeting of LIM and SH3 protein 1. Another study reported that the expression level of miR-206 is significantly lower in hepatocellular carcinoma (HCC) tissues, and miR-206 inhibits the growth of HCC cells through targeting cyclin-dependent kinase 9 (33). In the present study, it was observed that miR-206 is significantly downregulated in human ESCC tissues and cell lines, and the low expression level of miR-206 was closely correlated with key clinicopathological properties, as well as overall survival in ESCC patients. In addition, overexpression of miR-206 reduced ESCC cell proliferation and induced cell apoptosis in vitro. These data were consistent with the results of a previous study, demonstrating that miR-206 functions as a tumor suppressor in clear-cell renal cell carcinoma (34).

Several recent reports have proven that c-Met is overexpressed in several cancer types, including gastric and breast cancer (35–37). c-Met, a well-known oncogene, has been reported to promote cell proliferation and invasion (27). Of note, many studies have reported that the c-Met is regulated by miRNAs, such as miR-146a, miR-181a-5p and miR-101 (38–40). Furthermore, c-Met has been identified to be a direct target of miR-206 in lung cancer cells (41,42). In the present study, it was demonstrated that miR-206 directly bound to the 3′UTR of c-Met and negatively regulated its expression in ESCC cells. The restoration of c-Met expression inhibited the anti-tumor activities of miR-206 in ESCC cells.

It is well established that c-Met is an important regulator of the Akt/mTOR pathway (43–45), and activation of the phosphoinositide 3-kinase (PI3K)/mTOR signaling pathway has been reported to have a pivotal role in the progression of ESCC (46–48). For example, Liu et al (25) demonstrated that miR-206 inhibits head and neck squamous cell carcinoma progression via the AKT/mTOR pathway. Another study from Liu et al (49) reported that using LY294002, a PI3K specific inhibitor, significantly inhibits ESCC cell proliferation and migration. As previously mentioned, the present study demonstrated that c-Met may be a direct target of miR-206 in ESCC cells. Therefore, it was speculated that miR-206 affected the proliferation and apoptosis of ESCC by mediating the c-Met/AKT/mTOR signaling pathway. The expression levels of a number of c-Met/AKT/mTOR signaling pathway-associated proteins were examined, including p-c-Met, c-Met, p-AKT, AKT, p-mTOR and mTOR. The results demonstrated that the expression of these proteins was significantly inhibited, suggesting that miR-206 exerted its anti-tumor effects via the c-Met/Akt/mTOR pathway.

Although the present study supported the conclusion that miR-206 functioned as a suppressor of cell growth and as a prognostic marker for longer survival in ESCC, other target genes of miR-206 or other differentially expressed miRNAs identified in the present work should also be carefully tested for their significance in ESCC. In addition, the cohort for the survival analysis was small, and larger cohorts are required in order to verify the results of the present study.

In conclusion, the present study demonstrated that miR-206 was downregulated in ESCC and overexpression of miR-206 suppressed ESCC cell proliferation and induced cell apoptosis, potentially via the c-Met/AKT/mTOR signaling pathway. Overall, these findings suggested that miR-206 may be a novel target for ESCC treatment.