Te 13

Human esophageal epithelial cells (Het-1A) and ESCC cell lines (TE-13, KYSE140, EC9706, and KYSE30) were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco, USA) in a 37 °C incubator with 5% CO₂.

This study was approved by Ethics Committee of Zhengzhou University, and informed consent was obtained from each patient. A total of 62 EC patients with primary ESCC who took Neoadjuvant chemotherapy after esophagectomy in The First Affiliated Hospital of Zhengzhou University were recruited in this study. ESCC tissues and their paired adjacent normal esophageal epithelial tissues were collected and stored at − 80 °C. According to the National Comprehensive Cancer Network esophageal cancer guideline, the normal tissues were at least 5 cm away from the primary lesion.
Combined chemotherapy for the treatment of ESCC was cisplatin, 5-Fu and adriamycin, or cisplatin, 5-Fu and paclitaxel. According to Response Evaluation Criteria in Solid Tumors (RECIST) guideline, patients were divided into complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD) after complete chemotherapy. Patients with CR and PR were defned as responders, whereas those with SD and PD were defned as non-responders.
ESCC cell lines (TE-13, KYSE140, EC9706, KYSE30) and human esophageal epithelial cells (Het-1A) were purchased from Cell Bank of TypeCulture Collection of Chinese Academy of Sciences (Shanghai, China), and cultured in RPMI1640 supplemented with 10% fetal bovine serum (Gibco, USA) in 5% CO₂ incubator under 37 °C.

Five ESCC cell lines EC9706, Eca109, TE-13, TE-1 and TTN were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The five cell lines were cultured in medium and renewed every 24 h for subculture. The cell lines with the highest expression of ZFAS1 were screened out for subsequent experiments by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The cells were seeded in 6-well plates at the density of 3 × 10⁵ cells/well. When the cell confluence reached 80%, lipofectamine 2000 (Invitrogen, Carlsbad, California, USA) kit was used for transfection. Four μg target plasmid and ten μL lipofectamine 2000 was diluted by 250 μL serum-free Opti-MEM (Gibco, Carlsbad, California, USA) medium, respectively. After being placed for 20 min, the mixed solution was added to the culture well, and then cultured with 5% CO₂. After 6 h, it was replaced to complete culture medium, and the cells were collected after culturing for 48 h.