All patients provided written informed consent for the collection and use of their tissue samples. The present study was approved by the Ethics Review Committee of the Third People's Hospital of Linyi (Linyi, China). In total, 46 pairs of ESCC and adjacent-normal tissues were collected from the Department of Oncology at the Third People's Hospital of Linyi between April, 2016 and March, 2018. All tumors were pathologically confirmed as ESCC, and no other malignant tumors were identified. None of the patients had received neoadjuvant therapy (chemotherapy or radiotherapy) prior to surgery.

The StarBase database (starBase, v2.0, http://starbase.sysu.edu.cn/) was employed to identify potential miR-lncRNA binding partners. TargetScan (http://www.targetscan.org/vert_72/) was then used to determine the downstream target genes of these miRs.

Human ESCC cell lines (KYSE-30, KYSE-180, KYSE-150 and TE-5) and a normal esophageal epithelial cell line (Het-1A) were procured from the Shanghai Institute of Biochemistry and Cell Biology. All the cell lines were cultured in DMEM (HyClone; GE Healthcare life Sciences) containing 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (all Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C (5% CO₂). The media were replaced at 3-4-day intervals, and the cells were sub-cultured using the trypsinization method (0.25%; Amerecesco, Inc.).

The empty pcDNA vector (NC), pcDNA-LINC00473 (LINC00473), short hairpin (sh)RNA negative control (sh-NC), shRNAs targeting LINC00473 (sh-LINC00473), miR control (miR-NC), miR-497-5p mimic and miR-497-5p inhibitor were designed and constructed by Shanghai GenePharma Co., Ltd. KYSE-30 and TE-5 cells were seeded into a 24-well plate (3×10⁵ cells/well) and cultured for 24 h at 37°C (5% CO₂), prior to transfection using Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc.). All transfections were performed using a final concentration of 60 nM of miR-497-5p mimics and 100 nM of miR-497-5p inhibitor and sh-LINC00473. Reverse transcription-quantitative (RT-q)PCR was conducted to confirm transfection efficiency 24 h post-transfection, and the subsequent experiments were then performed.

Transfected ESCC cells were harvested in the logarithmic phase, and irradiated using a linear accelerator (Varian Medical Systems) at room temperature. The cells were exposed to various doses of radiation (0, 2, 4, 6 and 8 Gy) for 24-96 h prior to subsequent analysis. For the time-course experiment, cells were irradiated with X-rays at a dose of 6 Gy, and were then collected for immediate RT-qPCR analysis; RT-qPCR was conducted every 3 h within a 24 h period.

Total RNA was isolated from the ESCC tissues and cultured cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.); 1 μg total RNA was reverse transcribed into cDNA using the SuperScript™ First Strand cDNA System (Invitrogen; Thermo Fisher Scientific, Inc.), and qPCR was subsequently performed. SYBR-Green Premix Ex Taq II (Takara) and ABI 7500 real-time PCR system (Applied Biosystems) was applied for qPCR. The qPCR cycling conditions were as follows: 95°C for 2 min, then 40 cycles at the conditions of 95°C for 15 sec, 60°C for 15 sec, and 68°C for 20 sec. The relative expression levels of LINC00473 and miR-497-5p were calculated using the 2^−ΔΔCq method (31). The primers sequences were as follows: LINC00473 forward, 5′-GATGGAAAGGAGGGAAGG-3′ and reverse, 5′-CACAGTGGGTCCAGGGTT-3′; miR-497-5p forward, 5′-CCTTCAGCAGCACACTGTGG-3′ and reverse, 5′-CAGTGCAGGGTCCGAGGTAT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; and β-actin forward; 5′-TTCGAGCAAGAGATGGCCA-3′ and reverse, 5′-TACATGGTGGTGCCGCC-3′.

The ESCC tissues and cells were lysed with RIPA buffer (Beyotime Institute of Biotechnology) supplemented with protease inhibitors. Following high-speed centrifugation (10,000 × g, 4°C, 5 min), the supernatants were collected and the proteins were denatured in a water bath at 100°C for 10 min; the protein concentrations were determined using the bicinchoninic acid method. A total of 10 μl total protein extracts were loaded into each well, and were separated by SDS-PAGE using 12% gels, and then transferred to polyvinylidene fluoride membranes. The membrane was washed once in PBS with 0.2% Tween-20 (PBST) and then blocked with 5% non-fat milk for 1 h at room temperature. After washing in TBST solution, the membranes were incubated overnight with primary antibodies against CDC25A (cat. no. ab79252; Abcam, 1:1,000) and β-actin (cat. no. ab20272; Abcam; 1:100) on a shaking platform at 4°C. The membranes were then rinsed with TBS-T solution and incubated with goat anti-rabbit IgG H&L (cat. no. ab150077; Abcam; 1:1,000) at room temperature for 1 h. Following a final wash with TBS-T (3 times), signals were developed with Hypersensitive ECL Chemiluminescent Substrate (Hubei Biossci Biotechnology Co, Ltd.) and exposed to X-ray films.

The KYSE-30 and TE-5 cells were harvested in the logarithmic phase and adjusted to a density of 1×10⁴/ml; 100 μl cell suspension was then inoculated into the wells of a 96-well plate as appropriate, and cultured at 37°C for 24 h. Subsequently, 10 μl enhanced CCK-8 solution (Hubei Biossci Biotechnology Co, Ltd.) was added and the plate was returned to the incubator for a further 1 h. The absorbance (OD value) of each well was determined at 450 nm using a plate reader (Multiscan FC; Thermo Fisher Scientific, Inc.) at 24, 48, 72 and 96 h.

Transfected ESCC cells were seeded into 6-well plates at a density of 1×10³ cells/well and irradiated at a specified single dose (0, 2, 4, 6 or 8 Gy). The culture medium was then discarded and the cells were carefully washed twice with PBS. Following incubation at 37°C (5% CO₂) for 2 weeks, the cells were fixed with 10% methanol, stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA) for 15 min and dried at room temperature. Subsequently, the number of colonies was recorded under a light microscope (Olympus Corporation).

Wild-type (WT) or mutant-type (MUT) LINC00473 was subcloned into a pGL3 Basic vector (Promega Corporation). The cells were then seeded into a 24-well plate at a density of 5×10³ cells/well, and the Dual-Luciferase^® Reporter Assay System (Promega Corporation) was used to determine luciferase activity. miR-497-5p or miR-NC were co-transfected with WT or MUT reporter vectors into KYSE-30 and TE-5 cells, respectively, using Lipofectamine^® 3000. Luciferase activity was assessed at 48 h post-transfection. The results were normalized through comparison with Renilla luciferase activity.

All statistical analyzes were conducted using SPSS 20.0 (IBM Corp.), and all data are presented as the means ± standard deviation. Whether the data are normally distributed or not was examined using the Kolmogorov-Smirnov test. For normally distributed data, an unpaired or paired t-test was used to compare the data between 2 groups. Comparisons among ≥3 groups were conducted with one-way ANOVA. If the data exhibited significant differences, Tukey's post hoc test was then performed to compare the data between groups. For data that were not normally distributed, comparisons between 2 groups were performed by a paired sample Wilcoxon signed-rank test. Pearson's correlation coefficient was used to evaluate the correlation between the expression levels of the genes in the ESCC samples. A Chi-squared test was used to analyze the association between the expression of LINC00473 and the patient clinicopathological characteristics. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR was conducted to determine the association between the expression levels of LINC00473, miR-497-5p and CDC25A mRNA in 46 paired ESCC and adjacent-normal tissue samples. The expression levels of LINC00473 and CDC25A mRNA were significantly higher, while miR-497-5p expression was significantly lower in ESCC tissues, compared with those in the adjacent normal tissues (Fig. 1A-C). Additionally, western blot analysis was used to assess CDC25A protein expression in the ESCC and adjacent tissues of 5 randomly selected patient samples; the results indicated that CDC25A was also upregulated at the protein level (Fig. 1D). LINC00473 expression was also found to inversely correlate with that of miR-497-5p (Fig. 1E; r=-0.5102, P<0.001). Moreover, miR-497-5p expression negatively correlated with CDC25A expression (Fig. 1F; r=−0.3699, P<0.05), while LINC00473 expression positively correlated with CDC25A expression (Fig. 1G; r=0.3083, P<0.05). These data suggest a possible regulatory association among LINC00473, miR-497-5p and CDC25A.

The association between the expression of LINC00473 and the clinicopathological indexes of patients with ESCC was also analyzed. High expression levels of LINC00473 in tumor tissues were found to be significantly associated with local lymph node metastasis, a low degree of differentiation and a higher T stage in patients with ESCC; however, they were not associated with sex, age, tumor size or a history of smoking (Table I). These findings suggest that high expression of LINC00473 may be involved in the progression of ESCC.

RT-qPCR was used to quantify the expression levels of LINC00473 and miR-497-5p in 4 ESCC cell lines (KYSE-30, KYSE-150, KYSE-180 and TE-5). Compared with Het-1A cells, the expression of LINC00473 was significantly higher in the ESCC cell lines, while the expression of miR-497-5p was notably decreased (Fig. 2A and B). The KYSE-30 and TE-5 cells were irradiated with 6 Gy X-rays, and the expression of LINC00473 was detected every 3 h by RT-qPCR. The results revealed that the expression of LINC00473 in the irradiated KYSE-30 and TE-5 cells was significantly higher than that in the control group (0 Gy) (Fig. 2C and D), and that following irradiation, the expression of miR-497-5p was markedly suppressed (Fig. 2E and F). These results indicated that the expression of LINC00473 and miR-497-5p was inversely affected by X-ray irradiation.

The results of the aforementioned experiments illustrated that the expression of LINC00473 was upregulated in the irradiated ESCC cells. The expression level of LINC00473 was lowest in the KYSE-30 cells, and highest in the TE-5 cells; therefore, to further investigate the role of LINC00473 in the radiosensi-tivity of ESCC cells, KYSE-30 and TE-5 cells were selected to construct LINC00473 overexpression and knockdown models, respectively. The KYSE-30 cells were transfected with a LINC00473 overexpression plasmid, and the TE-5 cells with LINC00473 shRNA; RT-qPCR indicated the successful establishment of each model (Fig. 3A). CCK-8 assay was subsequently performed to assess the proliferative capacity of each cell line; the results revealed that LINC00473 over-expression significantly increased KYSE-30 cell proliferation at 48, 72 and 96 h, while LINC00473 knockdown inhibited the proliferation of TE-5 cells, demonstrating that LINC00473 enhanced the proliferative ability of the ESCC cells (Fig. 3B). A colony formation assay was then performed, and the results indicated that LINC00473 overexpression and knockdown increased and decreased the number of colony-forming units, respectively (Fig. 3C). Collectively, it was thus concluded that LINC00473 enhances the proliferative ability and reduces the sensitivity of ESCC cells to irradiation.

Bioinformatics analysis (starBase, v2.0, http://starbase.sysu.edu.cn/) predicted miR-497-5p to be a candidate target of LINC00473, and the potential binding site is illustrated in Fig. 4A. Using a dual-luciferase assay, following the transfection of miR-497 mimics into ESCC cells (Figs. S1 and S2), miR-497-5p overexpression was shown to markedly decrease the luciferase activity of pGL3-LINC00473-WT, whereas it did not significantly affect that of pGL3-LINC00473-MUT (Fig. 4B-D). The results suggested that of the three predicted binding fragments, the first and third had similar binding capacities, and the second exhibited the most statistically significant difference (Fig. 4B-D). In addition, the KYSE-30 and TE-5 cells were transfected with pcDNA-LINC00473 or sh-LINC00473. The results suggested that compared with the control group, the expression of miR-497-5p was notably inhibited by LINC00473 overexpression and increased by LINC00473 knockdown, respectively (Fig. 4E). Thus, LINC00473 can directly interact with miR-497-5p and negatively regulate its expression.

To investigate the potential mechanisms responsible for the radiosensitivity in ESCC cells induced by LINC00473, the KYSE-30 cells were transfected with pcDNA-NC, pcDNA-LINC00473 or pcDNA-LINC00473-miR-497-5p mimics; TE-5 cells were concurrently transfected with sh-NC, sh-LINC00473 or sh-LINC00473-miR-497-5p inhibitors (Figs. S1 and S2). The inhibitory effects of LINC00473 knockdown on ESCC cell viability were weakened by transfection with miR-497-5p inhibitors, while transfection with miR-497-5p mimics attenuated the promoting effects of LINC00473 on cell proliferation (Fig. 5A). Moreover, transfection with miR-497-5p mimics was shown to partially counteract the radioresistance of ESCC cells (2, 4, 6 and 8 Gy) induced by LINC00473, while transfection with miR-497-5p inhibitors exerted the opposite effect (Fig. 5B). Collectively, these data indicate that LINC00473 reduces the radiosensitivity of ESCC cells via miR-497-5p.

TargetScan predicted that CDC25A was one of the candidate target genes for miR-497-5p, and the speculative binding sites are displayed in Fig. 6A. The results of RT-qPCR and western blot analysis revealed that the mRNA and protein levels of CDC25A were notably decreased in the KYSE-30 and TE-5 cells following transfection with miR-497-5p mimics, respectively; on the other hand, miR-497-5p inhibitors induced the upregulation of CDC25A expression in ESCC cells at both the mRNA and protein levels (Fig. 6B and C). The results of the luciferase reporter assay indicated that miR-497-5p could specifically bind to the 3′UTR of CDC25A (Fig. 6D). The results of western blot analysis revealed that the expression of CDC25A was markedly elevated in the KYSE-30 and TE-5 cells following LINC00473 overexpression, but was reduced after LINC00473 was knocked down (Fig. 6E). Additionally, LINC00473 overexpression reversed the inhibitory effect on CDC25A expression induced by miR-497-5p (Fig. 6F). Furthermore, the expression level of CDC25A was markedly upregulated in the ESCC cell lines, compared with the Het-1A cells (Fig. 6G), and its expression in ESCC cells was induced by irradiation (Fig. 6H). Collectively, these data confirm that CDC25A is a downstream gene of miR-497-5p, and that its expression is directly and inversely regulated by miR-497-5p, though indirectly and positively modulated by LINC00473.