RNA extraction and RT-qPCR

Total RNA was extracted from HCT-116 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Isolated miRNAs were reverse transcribed using TransScript miRNA First-Strand cDNA Synthesis SuperMix (Beijing Transgen Biotech Co., Ltd.). Briefly, 5 µl total RNA, 1 µl TransScript^® miRNA RT Enzyme mix (Beijing Transgen Biotech Co., Ltd.), 10 µl 2X TS miRNA Reaction mix (Beijing Transgen Biotech Co., Ltd.) and 4 µl RNase-free water were mixed according to the manufacturer's instructions and incubated for 1 h at 37°C, followed by incubation for 5 sec at 85°C. Subsequently, 0.2 µM forward primer and 10 µl 2X TransScript^® Tip/Top Green qPCR SuperMix (Beijing Transgen Biotech Co., Ltd.) were mixed. The thermocycling conditions were: 94°C for 30 sec; followed by 45 cycles of 94°C for 5 sec, 60°C for 15 sec and 72°C for 15 sec; dissociation stage. The primer sequence for miR-27a-3p was 5′-TTCACAGTGGCTAAGTTCCGC-3′. mRNA was reverse transcribed with TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Beijing Transgen Biotech Co., Ltd.). qPCR was performed according to the manufacturer's protocol of the TransScript Top Green qPCR SuperMix (Beijing Transgen Biotech Co., Ltd.) using an iCycler thermal cycler (Bio-Rad Laboratories, Inc.). The thermocycling conditions were: 94°C for 30 sec; followed by 40 cycles of 94°C for 5 sec, 60°C for 15 sec and 72°C for 10 sec; return to room temperature. The comparative cycle threshold method (2^−ΔΔCq) (39) was used to conduct the relative quantification of target genes and miRNAs (10). Levels of miRNA and mRNA were normalized against U6 snRNA and GAPDH, respectively. The primer sequences were: BTG1 forward, 5′-AGCTGAACCTGTATCTGCGG-3′ and reverse, 5′-GAATTCCTGGTGCCAAAGGC-3′; U6 snRNA forward, 5′-ATTGGAACGATACAGAGAAGATT-3′ and reverse, 5′-GGAACGCTTCACGAATTTG-3′; and GAPDH forward, 5′-GGTGAAGGTCGGAGTCAACG-3′ and reverse, 5′-CAAAGTTGTCATGGA-3′. Transfection efficiency was determined by RT-qPCR.