Quantitative real-time polymerase chain reaction (RT-qPCR)

Total RNA from tissues and cell lines was extracted by using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Reverse transcription (RT) was conducted by using the Takara PrimeScript Kit (Takara) at 37°C for 15 min. Subsequently, the RT-quantitative (q)PCR assay was performed with the ViiATM 7 Real-Time PCR System (Thermo Fisher Scientific, Inc.) to detect the gene expression level. Relative gene expression was calculated using the 2^−ΔΔCq method (22). GAPDH and U6 were set as internal controls. The PCR amplification reaction was carried out using cDNA as template with the following conditions: 95°C for 10 min, 95°C for 15 sec, 62°C for 30 sec, and 72°C for 30 sec. Primers used for PCR are as follows: circCCDC66 forward, 5′-ACC TAC AAC CGG AAG CCA G-3′ and reverse, 5′-AGC AGT ACT GTT TCC TGA TGC-3′; miR-3140 forward, 5′-CTT CCA CTC GAC GTG CTG GAA GT-3′ and reverse, 5′-ACG GTC TCG TGC AGT CGT CAA CG-3′; Beclin1 forward, 5′-ACC GTG TCA CCA TCC AGG AA-3′ and reverse, 5′-GAA GCT GTT GGC ACT TTC TGT-3′; p62 forward, 5′-GCA GAA TGC CAT GGT TTC CC-3′ and reverse, 5′-GTG ATG GCT CCC CTT AC-3′; HIF1A forward, 5′-TAT GAG CCA GAA GAA CTT TTA GGC-3′ and reverse, 5′-CAC CTC TTT TGG CAA GCA TCC TG-3′; GAPDH forward, 5′-ACA ACT TTG GTA TCG TGG AAG G-3′ and reverse, 5′-GCC ATC ACG CCA CAG TT TC-3′; U6 forward, 5′-CTC GCT TCG GCA GCA CAT A-3′ and reverse, 5′-AAC GAT TCA CGA ATT TGC GT-3′.