Cell culture and quantitative real-time polymerase chain reaction (RT-qPCR)

The colorectal cancer cell line HCT116 and normal colon epithelial cell line NCM-460 were purchased from Fuheng Biotechnology (Shanghai, China). These cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, USA) with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin in an atmosphere containing 5% CO2 at 37° C.

For RT-qPCR assay, we first extracted total RNAs from cells using AG RNAex Pro reagent (Accurate Biology, AG21102). After then, these RNAs were reverse transcribed into cDNA using Evo M-MLV Kit (Accurate Biology, AG11705). The cDNA was eventually used for RT-qPCR analysis using SYBR Green Pro Taq HS Premix (Accurate Biology, AG11701). All the reactions were performed in StepOnePlus™ instrument. The 2^-ΔΔCt strategy was applied to compute the relative mRNA level of genes. Human GAPDH was utilized to normalize expression levels. The sequences of the primers were as follows:

Human GAPDH: 5’- GGAGCGAGATCCCTCCAAAAT GGCTGTTGTCATACTTCTCATGG-3’

COX17: 5’- GGTCGGGTCTCTGTTGACTT TTGCCGTTCTCCTCTCTCTC-3’

DLAT: 5’- CGGAACTCCACGAGTGACC CCCCGCCATACCCTGTAGT-3’