Abi 7500 fast pcr system

Total RNA in this study was isolated from cultured cells and tissues using Trizol reagent (Invitrogen, USA). Then, extracted RNAs were reverse-transcribed into cDNA by using Prime Script™ RT kit (Takara, Dalian, China). In addition, for miRNAs, MicroRNA Reverse Transcription Kit (Takara Biotechnology, Japan) were used to perform reverse transcription. The primers used in the current experiments were deigned and purchased from Geneseed Biomart (Guangzhou, China) (Table 1). qRT-PCR was carried out on ABI 7500 fast PCR System (Carlsbad, CA, USA) with a SYBR green PCR Master Mix (TOYOBO, Japan). GAPDH and U6 applied as internal references for mRNAs and miRNAs, respectively. The relative expressions were calculated with 2^–ΔΔCT method. For RNase R treatment, 1 unit of RNase R was added to digest 1 μg of RNA for 15 min at 37 °C.Table 1

Primers used in the present study.

Gene	Primer sequence
miR-187-3p	forward: 5′-CACAGGACCCGGGCG-3′
	reverse: 5′-CCGGCTGCAACACAAGAC-3′
miR-665	forward: 5′-CTCGCTTCGGCAGCACA-3′
	reverse: 5′-CAGTGCGTGTCGTGGAGT-3′
U6	forward: 5′-CTCGCTTCGGCAGCACA-3′
	reverse: 5′-AACGCTTCACGAATTTGCG′
hsa_circRNA_104348	forward: 5′-TCTGTGTGTCAAAGCAAGGC-3′
	reverse: 5′-AGATGCCACTGAATCACCCA-3′
RTKN2	forward: 5′-CTCATGGTGTGCAATGCTCG-3′
	reverse:5′-ACTGCAGATAGAGAAATGGACTT-3′
GAPDH	forward: 5′-CTCTGCTCCTCCTGTTCGAC-3′reverse: 5′- GCGCCCAATACGACCAAATC-3′

TRIzol reagent (Beyotime, Shanghai, China) was used to extract total RNA. BeyoFast™ SYBR Green One-Step qRT‒PCR Kit (Beyotime) was used to detect targeting gene according to the manual. In addition, for miRNAs, MicroRNA Reverse Transcription Kit (Takara Biotechnology, Japan) were used to perform reverse transcription. qRT-PCR was carried out on ABI 7500 fast PCR System (Carlsbad, CA, USA) with a SYBR green PCR Master Mix (TOYOBO, Japan). β-actin and U6 applied as internal references for mRNAs and miRNAs. The relative expressions were calculated with 2^–ΔΔCT method. Moreover, genomic DNA (gDNA) was extracted by a Universal Genomic DNA Purification Mini Spin Kit (Beyotime) based on the manufacturer’s protocol. All primers were synthesized by GenePharma (Shanghai, China) and are listed in Table S2.