Reverse transcription quantitative PCR (RT-qPCR) analysis

Total RNA was extracted as aforementioned, and reverse transcribed with SuperScript™ II Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. In brief, oligo(dT) primers (500 µg/ml), dNTP mix (10 mM each), RNA (1 µg) and ddH₂O were mixed, heated to 65°C for 5 min and quickly chilled on ice. Subsequently, 5X first-strand buffer, 0.1 M DTT and RNaseOUT (40 U/µl) were added, mixed and incubated at 42°C for 2 min. SuperScript II reverse transcriptase (200 U) were added, incubated at 42°C for 50 min and inactivated at 70°C for 15 min. For qPCR analysis, SYBR green (Bio-Rad Laboratories Inc.), target-specific primers (Table SI) and 5 ng cDNA template were used. The samples were analyzed on a ViiA 7 Real-Time-PCR system (Thermo Fisher Scientific, Inc.) using the following conditions: Hold stage at 50°C for 2 min; initial denaturation at 95°C for 10 min; followed by 40 cycles of 95°C for 15 sec, 63°C for 2 sec and 60°C for 1 min; melt curve analysis at 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. Data were normalized to the housekeeping genes TATA-box binding protein and β-actin, and displayed in relation to the wild-type cells or non-malignant tissue using the 2^−∆∆Cq method (16).