RNA extraction and reverse transcription-quantitative PCR (RT-qPCR)

Total RNA from cells and samples was extracted using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). The mRNA and miRNA were reverse-transcribed using the Prime Script RT Reagent kit and One Step Prime Script miRNA cDNA Synthesis kit, respectively, (both from Takara Bio, Inc.) following the manufacturer's guidelines and recommended thermocycling conditions. The cDNA was amplified using the SYBR Green (Takara Bio, Inc.) and Real-Time PCR (qPCR) System (QuantStudio3; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 95°C for 30 sec, 40 cycles at 95°C for 5 sec and 60°C for 30 sec, 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. U6 and ACTB were used as the internal controls for normalization and comparison. The 2^−ΔΔCq method (21) was used to calculate the expression level of the specific genes. The primers are presented in Table SIA.