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

Abstract

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.

Keywords: esophageal squamous cell carcinoma; microRNA‑206; c-Met; AKT/mTOR pathway.

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.

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.

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.

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.

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.