In total, two separate samples were collected (20 cases in total). The first group included 4 paired cases of ESCC and normal tissues (53-75 years; 3 males; 1 female), which were collected between May 2018 and July 2018 during resection of cancer via open surgery from the Department of Thoracic Surgery at Zhongda Hospital Affiliated to Southeast University (Nanjing, China). The second group included 16 pairs of ESCC tissues and corresponding normal tissue samples (50-75 years; 10 males; 6 females), which were collected between February 2019 and September 2019 after surgical operation and endoscopic submucosal dissection surgery at the Zhongda Hospital Affiliated to Southeast University (Nanjing, China). Healthy or normal esophageal epithelial tissue was defined as tissues that were ≥5 cm apart from the margin of the cancer tissues. All patients with ESCC had not received local or systemic treatment before operation. Excised ESCC tissue samples and corresponding healthy tissue samples were immediately frozen with liquid nitrogen and stored at 80°C. The study was approved by the Research Ethics Committee of the Southeast University (Nanjing, China; approval no. 2019ZDSYLL022-P01). All patients provided written informed consent.

Next-generation RNA sequencing assay was performed at Kangchen Biotechnology Co., Ltd. using Illumina HiSeq 4000 (Illumina, Inc.) to detect the mRNA and ncRNA expression profiles. Image processing and base recognition were performed using Solexa pipeline version 1.8 software (Off-Line Base Caller software). Then, the reference genome was compared using Hisat2 software (18) (version 2.1.0;, and a FPKM calculation at the gene and transcript levels was performed using R software 'Ballgown'. The differentially expressed lncRNAs and mRNAs were selected by fold change (FC)/P-value/false discovery rate (FDR) filtration (multiple ≥1.5, P<0.05 and FDR <0.05).

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