Ncm460

Four commonly used CRC cell lines (HT29, HCT116, SW480, SW620) and one human normal colonic epithelial cell line NCM460 were obtained from ATCC. HT29 was highly differentiated, HCT116 and SW480 were CRC cell lines in situ, while SW620 was advanced lymph node metastatic CRC cell. HT29 was routinely cultured in McCOy’s 5A medium (Gibco, Carlsbad, CA, USA), HCT116 was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (ibid.), while the other three cell lines were cultured in RPMI 1640 medium (ibid.), added with 10% (vol/vol) fetal bovine serum (FBS), and 1% penicillin/streptomycin in a humid atmosphere of 5% CO₂.
The human umbilical vein endothelial cells (HUVECs) were bought from ATCC and cultured in DMEM, added with 1% penicillin/streptomycin and 10% FBS in a humid incubator containing 5% CO₂ at 37 °C.

SW480, LS174T, RKO, LOVO cells (DMEM, 10% FBS), HT29, NCM460 (McCoy's 5A, 10% FBS), HCT116, and DLD1 (RPMI‐1640, 10% FBS) were purchased from ATCC and maintained at 37℃ with 5% CO₂.
All clinical samples utilized in this study, including primary CRC tissues and paired‐adjacent noncancerous colon tissues further than 5 cm, were collected from patients undergoing radical colon resection at the Department of Gastroenterology, Shenzhen Hospital, Southern Medical University (Guangdong, China). Fresh samples were frozen in liquid nitrogen immediately after resection and stored at −80℃. Samples were histologically stained with hematoxylin and eosin, and evaluated by experienced gastrointestinal pathologists for histological grade of cancers based on criteria set by the World Health Organization. Normal colorectal mucosa was defined as all straight, nonbranching crypts with histopathologically normal cells. All protocols were approved by the Ethic Committee of Southern Medical University (NYSZYYEC20190013) after obtaining patients’ informed consent. Samples details were summarized in Table S1.

Colon cancer cell lines (SW480, SW620 and HCT8) and normal human colon epithelial cell line (HIEC and NCM460) were obtained from ATCC (American Typical Culture Center). All cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (GIBCO), 100units/mL penicillin and 100μg/mL streptomycin in an incubator with 5% CO₂ at 37°C.

Human CRC cells (LoVo, RKO, SW480, HCT116, and HT-29) and normal colon mucosa cells (NCM460) purchased from ATCC were kept in RPMI-1640 with 10% FBS (Gibco) at 37°C. The pcDNA3.1-ADAMTS9-AS2, pcDNA3.1-FTO and pcDNA3.1-YTHDF2 plasmids, the small interfering RNAs (siRNAs) specific for BTG2 (si-BTG2) and ADAMTS9-AS2 (si-ADAMTS9-AS2), and short hairpin (sh) RNA for YTHDF2 (sh-YTHDF2) were obtained from GenePharma (Suzhou). Hsa-miR-27a-3p mimics/inhibitors/negative control (NC) were obtained from GenePharma. Lipo2000 was adopted for transfections.

Human CC cell lines (DLD1, HCT116, SW480, LoVo, Caco2 and HT29) and normal colon mucosal epithelial cell line (NCM460) were purchased from ATCC. All cells were cultured in DMEM supplemented with 10% FBS under standard culture conditions (5% CO₂, 37 °C). RNase R (Epicentre Technologies, USA) was used to degrade linear mRNA. In brief, we extracted RNAs from CC cells and split RNA to two parts: one for RNase R digestion and another for control with digestion buffer only. The samples were incubated at 37 °C for 30 min. Then the expression levels of circFNDC3B and FNDC3B were detected by quantitative real-time RT-PCR.