Mirna mimic

SW982 cells were cultured in DMEM high glucose medium (Hyclone, USA) supplemented with 10% FBS (Hyclone, USA) and 0.2% penicillin/streptomycin. Gain of miR-15/107 function was achieved by transfecting 10 nM miRNA mimic (Genepharma company, China) with Lipofectamine 3000 reagent (Invitrogen, USA) according to the manufacturer’s instruction. The scramble miRNA mimic served as negative control (NC). 48 h after cell transfection, CCK8 reagent was applied to the cells and incubated at 37 °C for 1 h. Optical density value was measured at 450 nm. The data was further normalized as percentage against NC group. Results for cell proliferation assay are represented as the mean ± SEM from the three independent cell experiments. Mann-Whitney test was used to analyze the statistical difference between the indicated group and NC group. P value less than 0.05 was considered as statistically significant.

Cells were transfected with the miRNA mimic (40 nM), siRNAs (80 nM) specific for hub genes or their corresponding negative controls (GenePharma, Shanghai, China) using a Lipofectamine 3000 kit (Thermo Fisher Scientific, Waltham, MA, United States) following the manufacturer’s instructions. The sequences of the siRNAs are shown in Supplementary Table S3.

Human fetal lung fibroblast (HFL1) cells and human embryonic kidney (HEK293T) cells were purchased from the Cell Resource Center of Shanghai Institutes for Life Sciences, Chinese Academy of Sciences. The HFL1 and HEK293T cells were cultured in F12K and DMEM medium containing a mixture of 10% FBS and 1% penicillin-streptomycin, respectively, and placed in a 37 °C incubator containing 5% CO₂. The growth of HFL1 cells was observed for a fixed period.
Information on the plasmids, siRNA, miRNA mimic, and probe sequence is provided in Supplementary Table S1. All plasmids, miRNA mimics, and siRNA were transfected with liposome 8000 (Beyotime Biotechnology, Beijing). pcDNA 3.1 (+) circSPON1 Mini, si-circSPON1, and miRNA mimic were synthesized by GenePharma. The administration doses for intracellular plasmid and siRNA transfection were according to Lipo 8000 instructions.

ATCC provided all cell lines (LO2, MHCC-97H, LM3, HepG2 and Huh-7) for this study. The cells were cultured under standard conditions in DMEM (Corning, NY, USA) and confirmed to be mycoplasma-free. A circMYBL2 overexpression vector was constructed by Hanheng Biotech (Shanghai, China). The construct used for RNAi-mediated knockdown of circMYBL2, the miRNA mimic, and the miRNA inhibitor were synthesized by GenePharma (Shanghai, China). Transfection of cells was carried out by Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA).

MiR-130b inhibitor (anti-M), miR-130b mimic, or the appropriate negative controls of miRNA inhibitor (anti-MC) and miRNA mimic were purchased from GenePharma (Shanghai, China). Anti-M and anti-MC were transfected at a final concentration of 50–100 nM in the cells using HiPerFect Transfection Reagent (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. Expression of PPARγ and VEGF-A were knocked down with small interfering RNA (siRNA) duplexes using Oligofectamine (Invitrogen, Carlsbad, CA). The target sequences for PPARγ and VEGF-A mRNA were shown in Table 2. Non-targeting siRNA pool (D-001206-13-05; Dharmacon, Fisher Scientific, Pittsburgh, PA) was used as a negative control. Cells were harvested 72 hours post transfection for analysis.Table 2

Target sequences for PPARγ and VEGF-A mRNA

Genes	Target sequences
PPARγ	5’-AAUAUGACCUGAAGCUCCAAGAAUAAG-3’
VEGF-A	5’-TGCTGTGAAGATGTACTCTATCTCGTGTTTTGGCCACTGACTGACACGAGATAGTACATCTTCA-3’