All ESCC and adjacent normal tissues used in the present study were collected in Dongnan Affiliated Hospital of Xiamen University (Zhangzhou, China). Written informed consent was obtained from all patients. Ethical approval for the collection and use of all samples was approved by the Ethics Committee of Dongnan Affiliated Hospital of Xiamen University. Fresh tissues were immersed in RNAlater (Qiagen GmbH, Hilden, Germany) in 30 min after resection and subsequently stored at −80°C for future use.

Human ESCC cell lines Eca109 and TE-1 were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). Eca109 and TE-1 were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 U/ml penicillin and 100 g/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.), at 37°C for 24 h in a humidified incubator containing 5% CO₂. For the restoration of miR-486-5p in ESCC tissues with endogenously downregulated miR-486-5p, synthesized miR-486-5p mimics (GenePharma Co., Ltd., Shanghai, China) was transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. The cells were trypsinized, and total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) 24 h after transfection.

Total RNA was extracted from 36 ESCC tissue samples and adjacent normal esophageal tissues, or from the trypsinized ESCC cell lines Eca109 and TE-1, using TRIzol reagent (Invitrogen), and purified using an RNeasy Maxi Kit (Qiagen GmbH) according to the manufacturer's protocol. To obtain the cDNA templates, 1 µg total RNA of each sample was used for reverse transcription using an miScript Reverse Transcription kit (Qiagen GmbH). This reaction was performed at 37°C for 60 min, then 95°C for 5 min. The qPCR reaction of miR-486-5p was performed on an ABI PRISM 7000 Fluorescent Quantitative PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using miScript SYBR Green PCR Kit (Qiagen GmbH). The 20 µl reaction mixture contained 1 µl cDNA template, 2 µl 10X miScript Universal Primer, 0.4 µl of each of the specific miRNA primers, 10 µl 2X QuantiTect SYBR Green PCR Master mix and 6.6 µl RNase-free water. Primer sequences were as follows: Forward, 5′-TCCTGTACTGAGCTGCCCCGAG-3′ [the reverse primer was provided by the miScript SYBR Green PCR Kit (Qiagen GmbH)] for miR-486-5p; and forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-ACGCTTCACGAATTTGCGT-3′, for U6 (U6 was used as an endogenous control in the present study). Amplification conditions were set as follows: 95°C for 2 min, followed by 95°C for 15 sec, 58°C 30 sec and 72°C for 30 sec, for 40 cycles. This experiment was repeated 3 times, with accompanying no cDNA and no reverse transcriptase controls. The expression of miR-486-5p was analyzed using the ΔΔCq method (17), normalizing to U6 expression.

A wound scratch assay was used to assess the migratory ability of Eca109 and TE-1 cells in vitro. Cells (~150,000) were seeded into a 12-well dish and transfected with miR-486-5p mimics (60 pmol) or the negative, scrambled control (60 pmol) (GenePharma Co., Ltd.) 24 h later. A sterile 200 µl pipette tip was used to scrape a clear line through the cell monolayer 5 h post-transfection. The cells were then rinsed three times with phosphate-buffered saline (PBS) and cultured in an incubator at 37°C. Images of the wound scratches were acquired with an inverted light microscope (DM IRB; Leica Microsystems GmbH, Wetzlar, Germany) at 0 and 24 h after the wounds were made. The migration distance (µm) was measured with a standard caliper and the experiments were performed in triplicate and analyzed by at least two observers.

The cell proliferation of Eca109 and TE-1 cells was determined with MTT assay kit (Sigma-Aldrich, St. Louis, MO, USA). To determine cell growth, ~5,000 cells were seeded into the wells of 96-well plates and transfected with miR-486-5p mimics (5 pmol) or the negative, scrambled control (5 pmol). MTT (20 µl; 5 mg/ml; Sigma-Aldrich) was added to each well at 0, 24, 48 and 72 h after transfection. Subsequent to incubation for 4 h, the MTT medium was removed and 150 µl dimethyl sulfoxide was added. After shaking for 15 min at room temperature, the optical density (OD) of each sample was determined with an Enzyme Immunoassay Instrument (Model 680 microplate reader; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at a wavelength of 490/630 nm.

For apoptosis assays, Eca109 and TE-1 cells were cultured in 6-well plates at 37°C to a confluence of ~65% and transfected with miR-486-5p mimics or a negative control. After 48 h of transfection, Eca109 and TE-1 cells were harvested and washed twice with cold PBS, then resuspended in 10 µl 1X binding buffer (Invitrogen). Annexin V-FITC (5 µl; Invitrogen) and 10 µl propidium iodide was added to each sample. According to the manufacturer's protocol, the fluorescence of stained cells was then assessed by flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) using 488 nm excitation within 30 min.

Statistical analysis was performed using the SPSS statistical software package (version 17.0; SPSS, Inc., Chicago, IL, USA). Statistical significance was determined by paired and Student's t-tests. P<0.05 and P<0.01 were considered to indicate a statistically significant difference.

RT-qPCR was used to determine expression levels of miR-486-5p in 35 paired ESCC tissues and adjacent normal tissues. The relative expression of miR-486-5p in the 35 paired ESCC tissues and adjacent normal tissues are shown in Fig. 1A. miR-486-5p expression in ESCC tissues was significantly downregulated compared with those of the paired adjacent normal tissues (P<0.05) (Fig. 1B).

To analyze the function of miR-486-5p in ESCC, miR-486-5p mimics and negative controls were transfected into the ESCC cell lines, Eca109 and TE-1. Images of cells transfected with Fam-labeled negative control were obtained 6 h after transfection. As shown in Fig. 2A, the transfection efficiency was ~80 and ~85% in Eca109 and TE-1 cells, respectively. Compared with the negative control, the relative expression levels of miR-486-5p in Eca109 and TE-1 cells transfected with miR-486-5p mimics were 94- and 103-fold, respectively (P<0.05; Fig. 2B). The results demonstrated that miR-486-5p mimics were effective in upregulating the expression of miR-486-5p.

The effects of overexpression of miR-486-5p on cell migration of ESCC cells in vitro was determined by the use of a wound scratch assay. As shown in Fig. 3, the wound widths of Eca109 and TE-1 cells transfected with miR-486-5p mimics were wider (P<0.05) compared with those of the negative control group at 24 h. Thus, it was indicated that upregulation of miR-486-5p inhibited the cell migration of ESCC cells.

The impact of miR-486-5p on cell proliferation in ESCC cells was analyzed using an MTT assay. The OD values of the miR-486-5p mimic and negative control groups were measured at 0, 24, 48 and 72 h after transfection. The results showed that the proliferation of Eca109 cells decreased by 9.09, 15.71 and 19.84% (all P<0.05) at the respective aforementioned time-points, while the proliferation of TE-1 cells decreased by 8.89, 14.47 and 19.17% (all P<0.05; Fig. 4). These results suggest that the upregulation of miR-486-5p by mimics suppressed proliferation of ESCC cells in vitro.

To determine the effects of miR-486-5p on ESCC cell apoptosis, flow cytometry was used to determine the apoptosis rates after transfection. As shown in Fig. 5, apoptosis rates of Eca109 cells transfected with miR-486-5p mimics and those of the negative control were 9.7 and 3.4%, respectively (P<0.01) 48 h after transfection. The apoptosis rates of TE-1 cells were 10.4 and 4.4%, respectively (P<0.01) subsequent to transfection (Fig. 5). Thus suggesting that the restoration of miR-486-5p expression levels induced ESCC cell apoptosis.