Real-time quantitative polymerase chain reaction (qRT-PCR)

Total RNA was isolated using the Fastgen200 RNA isolation system (Fastgen, Shanghai, China) and was reverse-transcribed into cDNA using the Fermentas RevertAidTM Kit (MBI Fermentas, Canada). The PCR conditions for E-cadherin, N-cadherin and Snail were 95 °C for 2 min, followed by 40 cycles of 95 °C for 0.5 min, 50 °C for 0.5 min and 72 °C for 0.5 min. The following primers were used: E-cadherin-F: 5’-GAA CGC ATT GCC ACA TAC AC-3’, E-cadherin-R: 5’-GAA TTC GGG CTT GTT GTC AT-3’, N-cadherin-F: 5’-TGT TTG ACT ATG AAG GCA GTG G-3’, N-cadherin-R: 5’-TCA GTC ATC ACC TCC ACC AT-3’, Snail-F: 5’-GCG AGC TGC AGG ACT CTA AT-3’, Snail-R: 5’-GGA CAG AGT CCC AGA TGA GC-3’, β-actin-F: 5’-CTT AGT TGC GTT ACA CCC TTT CTT G-3’, β-actin-R: 5’-CTG TCA CCT TCA CCG TTC CAG TTT-3’. β-actin was used as an internal control. The relative gene expression was calculated using the previously described 2^-ΔΔCt method.