Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from cancer cell lines and cancer tissues and normal tissues using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. qPCR was performed using the SYBRGreen detection RT-PCR system (Takara Bio, Inc.) with RT mix (cat. no., RR036B; Takara Bio, Inc.) and SYBRGreen (cat. no., 740703; Takara Bio, Inc.). Thermocycling conditions were as follows: Initial denaturation: 95°C for 5 sec; 40 cycles, 95°C for 5 sec of denaturation, 95°C for 35 sec and 60°C for 30 sec for annealing and elongation and 60°C for 30 sec for final extension Actin was applied for normalization. The following primer sequences were used for the qPCR: E-cadherin: Forward, 5′-AACTCCACCTCCTGAAGCTG-3′ and reverse, 5′-TTGCTTGACCTACTGCCAGA-3′; N-cadherin: Forward, 5′-TCCACCTACCTCCTGAAGCTG-3′ and reverse, 5′-TTGACCACCAGCTGTGAC-3′; Snail: Forward, 5′-ACCAACTACCAACACCAAG-3′ and reverse, 5′-TACCCACCAAGCTGTAG-3′; Slug: Forward, 5′-GGTATCATGGTCGGTATGGGT-3′ and reverse, 5′-TCTTTCAGCAGTGGTGGAGA-3′; Vimentin: Forward, 5′-TCCTACGTTGGTGATGAAGCT-3′ and reverse, 5′-TTCTCTTTCAGCAGTGGTGG-3′; Actin: Forward, 5′-TCATGGTCGGTATGGGTCAA-3′ and reverse, 5′-TCAGCAGTGGTGGAGAAAGA-3′; and RP11-480I12.5: Forward, 5′-ACGTTGGTGATGAAGCTCAA-3′ and reverse, 5′-GCAGTGGTGGAGAAAGAGTA-3′. All experiments were performed in triplicate. The 2^−ΔΔCq method was used to calculate relative RNA expression (15–17).