RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR)

All tissues and cells were treated with TRIzol^® reagent (Thermo Fisher Scientific) for total RNA isolation following the manufacturer’s instructions. RNA quality and concentration were determined by NanoDrop 2000 (Thermo Fisher Scientific, Rockford, IL, USA). Then, SuperScript IV VILO kit (Invitrogen, Carlsbad, CA, USA) was employed to synthesize cDNA, while for miRNA analysis, All-in-One™ First-Strand cDNA Synthesis Kit (GeneCopoeia, Guangzhou, China) was used. The ABI SYBR Green Master Mix (Invitrogen) was used for RT-qPCR. The 2^−ΔΔCT method was applied to calculate relative expression of circ_0006174, MACC1 mRNA and miR-138-5p. The primers sequences were shown as circ_0006174, forward: 5ʹ-CATCCATCACTCCAGCATCAG-3ʹ, reverse: 5ʹ-GGTCACCATAACCACCACAAAG-3ʹ; miR-138-5p, forward: 5ʹ-GCGAGCTGGTGTTGTGAATC-3ʹ, reverse: 5ʹ-AGTGCAGGGTCCGAGGTATT-3ʹ; MACC1, forward: 5ʹ-TTCTTTTGATTCCTCCGGTGA-3ʹ, reverse: 5ʹ-ACTCTGATGGGCATGTGCTG-3ʹ; glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward: 5ʹ-GGAGGGAGATCCCTCCAAAAT-3ʹ, reverse: 5ʹ-GGCTGTTGTCATACTTCTCATGG-3ʹ; U6 small nuclear RNA (U6), forward: 5ʹ-CTCGCTTCGGCAGCACA-3ʹ, reverse: 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ, 18S ribosomal ribonucleic acid (18S rRNA), forward: 5ʹ-GAGTGTAAGGACCCATCGGA-3ʹ, reverse: 5ʹ-CCTCCAATGGATCCTCGTTA-3ʹ. 18S rRNA and U6 were used as internal controls of circ_0006174. GAPDH and U6 were used as internal controls of MACC1 and miR-138-5p, respectively.