Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Total RNA was extracted from KYSE-410, KYSE-150, ECA-109, TE-1, and HET-1A cells by using TRIzol Reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. For circRNA analysis, cDNA was synthesized by using Prime Script^TM RT Master Mix (Takara, Japan), and for miRNA analysis, RNA was reverse transcribed into cDNA using the Mir-X miRNA First-Strand Synthesis Kit (Takara, Japan). The nucleus and cytoplasm were separated with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) following the manufacturer's instructions. qRT-PCR was conducted with SYBR® Premix Ex Taq™ (Takara) on a CFX96 Touch Deep Well detection system (Bio-Rad, USA) with the following primers: circ_0000700, 5'-CTGGCTGAACATATCCCTTGGC-3' (forward) and 5'-TCCAAGGGATATGTTCAGCCA-3' (reverse); U6, 5'-CTCGCTTCGGCAGCACA-3' (forward) and 5'-AACGCTTCACGAATTTGCGT-3' (reverse); and GAPDH, 5'-AGA AGGCTGGGGCTCATTTG-3' (forward) and 5'-AGGGGCCATCCACAGTCTTC-3' (reverse). The entire sequence of the mature miR-1229 miRNA sequence was used as the miRNA-specific 5' primer, and the 3' primer for qRT-PCR was the mRQ 3' primer supplied with the kit. GAPDH was used as the reference gene for circ_0000700, and U6 was selected as the internal reference for miR-1229. The relative expression of the aforementioned targets was assessed by the 2^-ΔΔCt method.