The present study was approved by the Fujian Medical University Union Hospital Institutional Review Board (No. 2014KY001). All of the patients signed informed consent prior to treatment, and all of the information was anonymized prior to its analysis. The pretreatment work-up and eligibility criteria, details of radiotherapy and chemotherapy, criteria for toxicity, and short-term response, follow-up and the statistical analysis of survival were presented in our previous study (Chen M. Q. et al., 2017).

Between July 2015 and March 2017, a total of 41 patients with ESCC who received RT were recruited. Tissue specimens obtained during pretreatment with esophagogastroduodenoscopy were histopathologically examined by two independent pathologists and were snap frozen in liquid nitrogen and then stored at −80°C until RNA extraction.

Tissue specimens were divided into a radiosensitive group (n = 23) and a radioresistant group (n = 18) based on short-term response to RT. The short-term responses to RT were classified as a clinically complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD) according to the Japanese Classification of Esophageal Cancer guidelines (Japan Esophageal Society, 2017). Of these, the CR and PR were termed radiosensitive group and the SD and PD were termed radioresistant group in the current study.

Microarray profiling was performed using three radiosensitive ESCC tumor tissues and three radioresistant ESCC tumor tissues. RNA extraction and sequential microarray hybridization were conducted by Biotechnology Company (Shanghai, China), and the detected human genome transcripts were obtained by the Human lncRNA array V6.0 (4x180 K; Agilent Technologies, Inc., Santa Clara, CA, USA). Bioinformatics analysis was performed using GeneSpring Software to obtain differentially expressed lncRNAs correlated with ESCC radiosensitivity.

The ESCC cell line Eca109 was obtained from Chinese Academy of Sciences (Beijing, China). The corresponding radioresistant cells (Eca109R) were established from the parental cell line Eca109 by stepwise X-ray irradiation at 30 Gy in three fractions (10 Gy per fraction) (Da et al., 2017). Cells were cultured in RPMI-1640 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) with 10% (v/v) fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin; HyClone) in an atmosphere of 95% air/ 5% CO₂ at 37°C.

Total RNAs from either tissue samples or cultured cells were extracted with TRIzol reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The RNA concentration and quality were measured using a NanoDrop ND-2000 spectrophotometer which measured the absorbance at 260 and 280 nm. Samples with an A₂₆₀:A₂₈₀ ratio ≥2.0 were selected for further analysis.

First strand cDNA for the potential lncRNAs and putative micro (mi)-RNA were synthesized using the PrimeScript^TM RT reagent kit with gDNA Eraser (Takara Biotechnology, Co., Ltd., Dalian, China) according to the manufacturer's protocol. Briefly, 1 μg total RNA, 2 μl 5X gDNA Eraser Buffer, 1 μl gDNA Eraser and RNase Free dH₂O, were combined in a total reaction volume of 10 μl and incubated at 42°C for 2 min to eliminate the genomic DNA. A total of 10 μl of the RT reaction mixture (consisting of 4 μl 5X PrimeScript Buffer 2, 1 μl PrimeScript RT Enzyme Mix 1, 1 μl RT Primer Mix, and 4 μl RNase Free dH₂O) was then added, and the mixture was incubated at 37°C for 15 min, followed by 85°C for 5 s to generate the cDNA.

The expression of the potential lncRNAs in the radiosensitive tumor tissues, compared with the radioresistant tumor tissues, was quantified using SYBR^Ⓡ Premix Ex Taq (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions on the ABI 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Briefly, the 20 μl reaction mixtures were incubated at 95°C for 30 s for the initial denaturation, followed by 40 cycles at 95°C for 5 s and 60°C for 34 s. The expression levels of lncRNAs were calculated using the ΔCt method, where ΔCt = Ct _target -Ct _reference, a smaller ΔCt value indicates a greater expression. The relative expression of lncRNAs was analyzed using the 2^−ΔΔCt method (Livak and Schmittgen, 2001); data was normalized to the endogenous control GAPDH. Each sample was examined in triplicate. The primers and oligonucleotides of the plasmid were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.), the sequences are presented in Table 1. The aberrant lncRNA that had the greatest sensitivity and specificity for predicting ESCC radiosensitivity (in radiosensitive and radioresistant tissues), as identified by receiver operating characteristic (ROC) curves, and was associated with survival, was identified as the candidate lncRNA for further study.

Small interfering RNA (siRNA) specifically targeting candidate lncRNA (si-candidate-lncRNA) and putative-miRNA, negative control (NC) si-candidate-lncRNA and si-putative-miRNA, candidate-lncRNA mimic, putative-miRNA mimic, and the inhibitor control were constructed by Nanjing Dongji Biotechnology Company (Nanjing, China). Ectopic expression of the candidate lncRNA was achieved by introducing the candidate lncRNA sequence into a pcDNA3.1 vector (Thermo Fisher Scientific, Inc.). Eca109/Eca109R cells were seeded into 6-well plates at a density of 1 × 10⁶ cells/well and cultured overnight prior to transfection. Then, transient transfection with oligonucleotides or plasmids into Eca109/Eca109R cells was performed using Lipofectamine 2000^TM (Thermo Fisher Scientific, Inc.). Cells were harvested 48 h post-transfection for subsequent analysis. PCR was used to validate the efficacy of Eca109/Eca109R cell transfection with si-candidate-lncRNA and candidate-lncRNA-mimic.

Protein samples from tissues or cells were subjected to 10% SDS-PAGE and transferred to PVDF membranes. Following blocking in 5% skim milk for 2 h, the membranes were incubated overnight at 4°C with the primary antibodies against P-glycoprotein (P-gp; 1:1,000), glutathione S-transferase π (GST-π; 1:500), ATM (1:750), mTOR (1:1,000), and β-actin (1:5,000) purchased from Zen Bioscience Biotechnology, Inc. (Chengdu, China), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies for 2 h (1:5,000). The antigen-antibody complexes were visualized using chemiluminescence.

Radiosensitivity was assessed by 4-5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. ECA109/ECA109R cells (5,000/well) were incubated for 48 h prior to exposure to various doses of radiation (0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy, and 10 Gy). Subsequently, 10 μl of 5 mg/mL MTT was added to each well for a further 3 h, followed by the addition of 150 μl DMSO to dissolve the generated formazan crystals. The absorbance at a wavelength of 570 nm was detected using a microplate reader.

ECA109/ECA109R cells (5,000/well) were incubated for 48 h prior to exposure to various doses of radiation (0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy, and 10 Gy). The ratio of apoptotic cells was detected using an Annexin V-FITC Apoptosis Detection Kit (BD Bioscience, Franklin Lakes, NJ, USA) and analyzed using a BD Calibur flow cytometer with CellQuest software (BD Biosciences).

The potential target genes downstream of the candidate lncRNA were predicted using Starbase 2.0 software (http://starbase.sysu.edu.cn/starbase2/index.php) and the TargetScan (www.targetscan.org/vert_71/) database.

The full fragments of the candidate lncRNA or its mutant containing the putative miRNA-binding sites were synthesized and cloned downstream of the firefly luciferase gene in pGL3 plasmids (Promega Corporation, Madison, WI, USA), and were termed the pGL3-candidate lncRNA-wild type (Wt) and pGL3-candidate lncRNA-mutant (Mut). Eca109 and Eca109R cells were maintained in 96-well plates and co-transfected with 400 ng of the constructed luciferase reporter plasmids, 50 ng of Renilla luciferase reporter vector and 50 nM of the putative miRNA mimic, miR-con, or putative miRNA-vector using Lipofectamine 3000^TM (Thermo Fisher Scientific, Inc.). Cells were harvested at 48 h after transfection, and luciferase activity was determined using a Dual Luciferase Reporter Assay Kit (Promega Corporation). Renilla luciferase activities were used as the internal control for the normalization of firefly luciferase activity.

The animal experiments were approved by the Animal Care and Use Committee of Fujian Medical University Union Hospital and were performed in accordance with the Institutional Guide for the Care And Use Of Laboratory Animals. Lentiviral vector [Lenti-short hairpin (sh)-candidate lncRNA] for stable silenced expression of the candidate lncRNA was obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China) and transfected into Eca109/Eca109R cells. The success of transfection was detected by PCR and the survival of the cells was determined by an MTT assay. Then, equal numbers of siRNA-candidate lncRNA-transfected Eca109, NC and control cells were implanted into 8-week old nude mice (n = 5 per group; Model Animal Research Center of Nanjing University) by subcutaneous injection.

At two weeks after the injection (to allow for tumor growth), the tumors were irradiated by X-ray at 10 Gy. Tumor size was measured every 3 days with a caliper, and tumor volume was calculated according to the following formula: Volume = (length x width²)/2. All mice were sacrificed on day 42 after inoculation. The resected tumor masses were harvested for subsequent weight measurements. A growth curve was constructed to determine tumor radiosensitivity and the effect of the siRNA of the candidate lncRNA on tumorigenicity in nude mice was analyzed.

The overall survival data was analyzed using SPSS software 23.0 (IBM Corp., Armonk, NY, USA). Survival curves were established through the Kaplan-Meier method and compared by a log rank test.

A multivariable analysis of patient demographic and clinical parameters (gender, age, ECOG score, tumor location, clinical T and N stages, the radiotherapy doses for GTV and CTV, and the tumor response to treatment) was performed using the Cox proportional hazards model.

Experimental data are presented as x¯±s from independent experiments performed in triplicate. For comparisons, paired or independent Student's t-tests, Chi-square tests or ANOVA with post hoc tests (Tukey's) were performed. ROC curves were used for selecting an optimal cut-off point for each test and for comparing the accuracy of diagnostic tests. Two-tailed P < 0.05 (^*P < 0.05, ^**P < 0.01, ^***P < 0.001) was considered to indicate a statistically significant difference.

Between July 2015 and March 2017, a total of 41 ESCC patients treated with RT combined with chemotherapy were enrolled in the present study. After RT, a total of 4 patients achieved CR, 19 patients reached PR, 9 patients maintained SD and 9 cases had PD. There were no significant differences between radiosensitive (4 CR and 19 PR) and radioresistant (9 SD and 9 PD) patients regarding the distributions of gender, age, ECOG score, tumor location, and clinical stage (Table 2).

A total of 113 aberrantly expressed lncRNAs were identified in the microarray analysis using three radiosensitive ESCC tumor tissues and three radioresistant ESCC tumor tissues, of which 71 lncRNA transcripts were upregulated (fold change >2, P < 0.05) and 42 lncRNA transcripts were downregulated (fold change < 0.5, P < 0.05) in the radiosensitive ESCC tumor tissues when compared with the radioresistant ESCC tumor tissues. The lncRNAs CASC2, FAM201A, DLEU2, DLX6-AS1, and MCF2L-AS1 were considered to be the potential lncRNAs related to radiosensitivity when analyzed using GeneSpring Software 12.6 (Agilent Technologies, Inc.) (Figure 1A, Supplementary File 1).

Tumor tissues from the remaining 35 enrolled patients (20 radiosensitive patients and 15 radioresistant patients, respectively) were collected to detect the expression of the lncRNAs CASC2, FAM201A, DLEU2, DLX6-AS1, and MCF2L-AS1 by RT-qPCR. The results revealed that the differential expression of CASC2, FAM201A, and DLX6-AS1 between the radioresistant and radiosensitive groups were significantly different, while the difference in the DLEU2 and MCF2L-AS1 expressions were not significantly different when comparing the groups (Figure 1B; Supplementary File 2).

Based on above data, the ROC curve of the lncRNAs CASC2, FAM201A, and DLX6-AS1 was applied to identify the lncRNA that was the most correlated to radiosensitivity and survival using the area under curve (AUC) were 0.783 (95%CI: 0.609–957, P = 0.005), 0.817 (95%CI: 0.673–960, P = 0.002), and 0.340 (95%CI: 0.150–530, P = 0.110); respectively. Compared with the lncRNA DLX6-AS1, FAM201A, and CASC2 yielded a superior AUC with specificity and sensitivity for distinguishing radiosensitive ESCC tumor tissues from radioresistant ESCC tumor tissues (Figure 2A).

CASC2 was associated with short-term response to RT but not with survival, while FAM201A was correlated with both the short-term response and survival (Figures 2B,C). This indicated that FAM201A, as opposed to CASC2, may be a suitable biomarker of ESCC treated with RT.

To analyze whether FAM201A functions as a biomarker for radiosensitivity and survival in ESCC or not, the maximum Youden index method (Fluss et al., 2005) was performed to establish the cutoff value of FAM201A in the ROC curve. A total of 22 patients were termed as FAM201A-low with an average ΔCt expression value of 6.155, whereas, the remaining 13 patients, named the FAM201A-high expression group, had an average ΔCt expression value of 8.437 (Supplementary File 3).

Compared with the FAM201A-low group, the FAM201A-high group exhibited a poorer short-term response to RT and lower survival time. However, neither high or low FAM201A expression was correlated with tumor stage, regardless of whether it was T or N stage (Table 3). Furthermore, univariate and multivariate analysis indicated that FAM201A was the only independent risk factor for survival (OR, 0.642; 95% CI, 0.4668–0.885; P = 0.007). These data suggested that FAM201A could be a robust molecular marker for predicting RT sensitivity and survival in patients with ESCC.

Based on the above results, the effects of FAM201A regulated radiosensitivity in ESCC cancer cells were further explored by performing an X-ray irradiation experiment using Eca109/Eca109R cells transfected with si-FAM201A and FAM201A-mimic (Figures 3A,B; Supplementary File 4).

The results revealed that the survival rates of both Eca109 and Eca109R cells decreased with the increasing X-ray irradiation dose, and the percentage of apoptotic cells in each line increased with the increasing X-ray irradiation dose (Figures 3C,D; Supplementary File 4). The decrease in survival was more pronounced with the increase in X-ray irradiation dose in ECA109 cells when compared with ECA109R cells, demonstrating that the Eca109R cells were more resistant to X-ray irradiation.

In Eca109 cells, when compared with the control cells, FAM201A-mimic exhibited a significant promotion in cell proliferation, while si-FAM201A exhibited a significant increase in proliferation inhibition, indicating that for Eca109 cells, upregulated FAM201A expression likely resulted in cell radioresistance to X-rays (Figure 3E; Supplementary File 4).

In Eca109R cells, when compared with the control cells, si-FAM201A exhibited a significant inhibition of cell proliferation, while FAM201A-mimic did not exhibit the increased cell proliferation that was observed in ECA109 cells, indicating that the expression level of FAM201A in Eca109R cells was already at a high level, and thus, further elevation of FAM201A expression was not possible to enhance its radioresistance. These results indicated that, whether in cases of intrinsic or acquired radioresistance, si-FAM201A may enhance ESCC cell radiosensitivity, which may therefore be a novel effective target strategy for sensitizing ESCC to radiotherapy (Figure 3F; Supplementary File 4).

To confirm the efficacy of si-FAM201A on radiosensitivity in vivo, a xenograft tumor mouse model was established. A total of 15 mice with similar weights and dates of birth were selected in the present study (male: female = 8:7). When compared with the control groups, FAM201A knockdown (sh-FAM201A) significantly blocked tumor growth (decreased tumor volume and weight), suggesting that the silenced FAM201A expression enhanced radiosensitivity, thereby confirming that FAM201A could induce radiosensitivity in vivo (Figures 3G,H; Supplementary File 4).

Using Starbase 2.0, miR-101 and miR-590 were predicted to have complementary base pairings with FAM201A. Accordingly, luciferase reporter vectors containing the Wt or a Mut FAM201A binding site were established and co-transfected with miR-101 into Eca109 cells. The same process was performed for miR-590.

The results demonstrated that the ectopic expression of miR-101 was markedly suppressed by co-transfection with the FAM201A mutant sequence in the Eca109 cell luciferase activity reporter assay. However, neither pGL3-FAM201A-Wt reporter nor pGL3-FAM201A-Mut transfection in Eca109 cells affected miR-590 expression (Figures 4A,B; Supplementary File 5).

To further evaluate the regulatory relationship between FAM201A and miR-101, Eca109 cells were transfected with si-FAM201A and FAM201A-mimic sequences and matched controls. The results revealed that miR-101 expression was significantly downregulated in FAM201A-mimic Eca109/Eca109R cells, and was notably upregulated in si-FAM201A-transfected Eca109/Eca109R cells (Figures 4C,D). Taken together, these results indicated that FAM201A suppressed the expression of miR-101 (Supplementary File 6).

Using TargetScan, ATM and mTOR were predicted to be the downstream targets of miR-101. In Eca109/Eca109R cells, the expression of ATM and mTOR was increased while that of miR-101 was decreased in FAM201A-mimic cells when compared with control cells. When FAM201A expression was decreased, the expression of ATM and mTOR was downregulated while that of miR-101 was increased. Compared with non-irradiated cells, the expression of ATM and mTOR increased after X-ray irradiation. Western blotting confirmed the results of PCR (Figure 5; Supplementary File 6).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.