All ESCC tissues and adjacent normal tissues used in the present study were collected from Dongnan Affiliated Hospital of Xiamen University (Zhangzhou, China) between February, 2013 and December, 2014. Written informed consent was obtained from all patients. The collection and use of these samples was approved by the Ethics Committee of Dongnan Affiliated Hospital of Xiamen University. Fresh ESCC and adjacent normal tissues were immersed in RNAlater (Qiagen GmbH, Hilden, Germany) in 30 min following resection, and subsequently stored at −80°C.

According to the manufacturer's protocol, total RNA was extracted with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and purified with RNeasy Maxi kit (Qiagen GmbH). Total RNA (1 µg of each sample) was used for RT using miScript RT kit (Qiagen GmbH) to obtain the complementary DNA (cDNA) templates. qPCR reaction of miR-193a-3p was performed in an ABI PRISM^® 7000 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using miScript SYBR Green PCR kit (Qiagen GmbH). U6 was used as an endogenous control. The 20 µl reaction mixture contained 2X QuantiTect SYBR Green PCR Master mix (10 µl), 10X miScript Universal Primer (2 µl), specific microRNA primer (0.4 µl), cDNA template (1 µl) and RNase-free water. The forward primer of miR-193a-3p was 5′-AACTGGCCTACAAAGTCCCAGT-3′ and the reverse primer was provided in the miScript SYBR Green PCR kit. The forward primer and reverse primers of U6 were 5′-CTCGCTTCGGCAGCACA-3′ and 5′-ACGCTTCACGAATTTGCGT-3′, respectively. Amplification conditions were set as: 95°C for 2 min, followed by 40 cycles of 94°C for 15 sec, 58°C for 30 sec and 72°C for 30 sec.

The human ESCC cell lines Eca109 and TE-1 used in the present study were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China), and were cultured in RPMI 1640 (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 g/ml streptomycin at 37°C in a humidified incubator containing 5% CO₂. For the downregulation of miR-193a-3p expression, a synthesized miR-193a-3p inhibitor (Shanghai GenePharma Co., Ltd., Shanghai, China) was transfected into the cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The transfection efficiency and changes in miR-193a-3p expression were determined by fluorescence microscopy and RT-qPCR, respectively.

The migratory ability of ESCC cells (Eca109 and TE-1) in vitro was assessed by wound scratch assay. Approximately 150,000 cells were seeded in a 12-well dish and were transfected with miR-193a-3p inhibitor (60 pmol) or negative control (60 pmol) with Lipofectamine 2000 24 h later. After 5 h of transfection, a vertical wound was created with the tip of a sterile 10-µl pipette, and markers were assigned to enable the observation of cells in the correct location. The cells were then rinsed three times with phosphate-buffered saline (PBS) and incubated at 37°C. Images of the wound scratches were captured with a digital camera system at 0 and 24 h after the wounds were made at the same location. Wound widths (µm) were measured with a standard caliper, and the experiments were performed in triplicate and analyzed by at least two observers.

Cell proliferation of ESCC lines (Eca109 and TE-1) was assessed with an MTT assay kit (Sigma-Aldrich, St. Louis, MO, USA). Approximately 5,000 cells were seeded into 96-well plates and transfected with 5 pmol of miR-193a-3p inhibitor or negative control. Next, 20 µl of MTT (5 mg/ml; Sigma-Aldrich) was added to the culture medium of each well at 0, 24, 48 and 72 h post-transfection. After incubation for 4 h, the MTT medium was removed, and 150 µl of dimethyl sulfoxide was added. After shaking for 15 min at room temperature, the optical density (OD) of each sample was assessed with an enzyme-linked immunosorbent assay instrument (model 680; Bio-Rad Rad Laboratories, Inc., Hercules, CA, USA) at a wavelength of 490/630 nm.

For apoptosis assay, ESCC cells (Eca109 and TE-1) were cultured in 6-well plates at 37°C and transfected with miR-193a-3p inhibitor or negative control at a confluence of ~65%. After 48 h of transfection, cells were harvested and washed twice with cold PBS, resuspended in 10 µl 1X binding buffer (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, 5 µl of Annexin V-fluorescein isothiocyanate (Invitrogen; Thermo Fisher Scientific, Inc.) and 10 µl of propidium iodide were added to each sample. The fluorescence of the stained cells was then analyzed by flow cytometry (Beckman Coulter, Inc., Brea, CA, USA) using an excitation wavelength of 488 nm within 30 min of staining, according to the manufacturer's protocol.

Statistical analysis was conducted with SPSS 17.0 statistical software package (SPSS, Inc., Chicago, IL, USA). Statistical significance was determined with paired t-test and Student's t test. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR was used to determine the expression of miR-193a-3p in 36 paired ESCC tissues and adjacent normal tissues. The relative expression of miR-193a-3p is represented in Fig. 1A. As shown in Fig. 1B, the miR-193a-3p expression in ESCC tissues was significantly higher than that in adjacent normal tissues (P=0.0153).

To determine the function of miR-193a-3p in ESCC, miR-193a-3p inhibitor and negative control were transfected into the ESCC cell lines Eca109 and TE-1. Images of the cells transfected with fluorescein amidite-labeled negative control were obtained at 6 h post-transfection, and revealed that the transfection efficiency was ~85 and 80% in Eca109 and TE-1 cells, respectively (Fig. 2A). Compared with the negative control, the relative expression of miR-193a-3p in Eca109 and TE-1 cells transfected with miR-193a-3p inhibitor was 5.5 and 7.3%, respectively (Fig. 2B). These results suggested that the miR-193a-3p inhibitor used in the present study was able to effectively downregulate the expression of miR-193a-3p.

Wound scratch assay was performed to determine the effects of miR-193a-3p on ESCC cell migration in vitro. Compared with the negative control group, the wound widths of Eca109 and TE-1 cells transfected with miR-193a-3p inhibitor were significantly wider (P=0.0322 and 0.0306, respectively; Fig. 3), which indicated that downregulation of miR-193a-3p inhibited the migration of ESCC cells (Fig. 3).

The impact of miR-193a-3p on cell proliferation in ESCC was analyzed by MTT assay. The OD values of miR-193a-3p inhibitor group and negative control group were measured at 0, 24, 48 and 72 h post-transfection. The results indicated that the proliferation of Eca109 cells decreased by 6.45% (P=0.0318), 10.63% (P=0.0131) and 14.19% (P=0.0019), while the proliferation of TE-1 cells decreased by 6.78% (P=0.0356), 11.51% (P=0.0152) and 14.72% (P=0.0025), at 24, 48 and 72 h post-transfection, respectively (Fig. 4). This indicated that downregulation of miR-193a-3p suppressed the proliferation of ESCC cells in vitro.

To determine the function of miR-193a-3p on ESCC cell apoptosis, flow cytometry was performed to detect the apoptosis rates upon transfection. As represented in Fig. 5, the apoptosis rates of Eca109 cells transfected with miR-193a-3p inhibitor and negative control were 7.2 and 2.7% (P=0.0121), respectively, following transfection for 48 h. The apoptosis rates of TE-1 cells were 6.6 and 2.8% (P=0.0154) upon transfection with miR-193a-3p inhibitor and negative control, respectively. These results demonstrated that inhibition of miR-193a-3p induced ESCC cell apoptosis.