Mirna first strand synthesis kit

Trizol reagent (Invitrogen) was utilized to obtain total RNA from melanoma tissues and cells. Then a HiScript III First Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) or a miRNA First Strand Synthesis Kit (Vazyme) was used to conduct the reverse transcription reaction. Next, the amplification reaction was performed using SYBR Mix (Vazyme) and a CFX96 Touch Deep Well Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The relative expression was analyzed using the 2^−ΔΔCt method and standardized by GAPDH or U6. The primer sequences were as follows: circ_0084043, F: 5′-TTCTAGACAGCCGGGGAGTG-3′ and R: 5′-CCAAAACCTTTCTTTCTTGATGGGA-3′; miR-31, F: 5′-GCCGCAGGCAAGATGCTGGC-3′ and R: 5′-CAGTGCAGGGTCCGAGGT-3′; KLF3, F: 5′-TGTCTCAGTGTCATACCCATCT-3′ and R: 5′-CCTTCTGGGGTCTGAAAGAACTT-3′; GAPDH, F: 5′-ACCACAGTCCATGCCATCAC-3′ and R: 5′-TCCACCACCCTGTTGCTGTA-3′; U6, F: 5′-GCGCGTCGTGAAGCGTTC-3′ and R: 5′-GTGCAGGGTCCGAGGT-3′.

TRIzol™ reagent (#15596026; Thermo Fisher) was used to prepare cell lysates as per the guidebook. A NanoDrop spectrophotometer was applied to detect RNA concentration. cDNA synthesis reagents (Vazyme, Jiangsu, China) and miRNA first strand synthesis kit (Vazyme) were used for reverse transcription. Then, diluted cDNA was subjected to qRT-PCR analysis on a qRT-PCR system (Bio-Rad, Hercules, CA, USA) with SYBR qPCR Master Mix (Vazyme). The expression of circ_0113656, miR-188-3p, and IFG2 was normalized to GAPDH or U6 by the 2^−∆∆Ct method. Random primers and oligo(dT)₁₈ primers were utilized to identify the circular structure of circ_0113656. Besides, 1 μg of RNA was exposed to RNase R (Xiyuan Biotech, Shanghai, China) at 37°C for 20 min to analyze the circRNA structure. Primers used for amplification are listed in Table 1.

Total RNA was extracted by TRIzol reagent (Invitrogen) from NRCMs and heart tissues following the manufacturer’s instructions. The concentrations of RNA were detected by NanoDrop ND-1000 spectrophotometer (Thermo Scientific). Then, RNA was reverse-transcribed into cDNA using the HiScript QRT Super Mix (Vazyme) and miRNA First-Strand Synthesis Kit (Vazyme) according to the manufacturer’s protocol. The real-time polymerase chain reaction was carried out using IQ SYBR Green Supermix (Bio-Rad) and the Prism 7500 SDS (Applied Biosystems). Finally, the relative expression value of genes was calculated using the 2^−ΔΔCt method. U6 was used as the internal control of miR-221/222, and β-actin was used as the internal control for the other genes. The primer sequences (Sangon Biotech) of the genes are shown in Additional file 1: Tables S3 and S4.