R0531

293T, SW480 and NCM460 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, SH30022.01, Hyclone, China), whereas LoVo, RKO, DLD-1 and HCT116 cells were cultured in Roswell Park Memorial Institute Medium 1640 (RPMI 1640, SH30809.01, Hyclone) supplemented with 10% fetal bovine serum (04-001, Biological Industries, Israel) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin, and SV30010, Hyclone), in a humidified atmosphere containing 5% CO₂ at 37 °C (normal culture conditions). Cell transfections were carried out by using Lipofectamine 2000 (11668019, Invitrogen, USA) or R0531 (Thermo Fisher Scientific, USA) according to the manufacturer's instructions.

For luciferase assays, 2 × 10⁴ cells/well were seeded in 48-well plate and transfected with the pmirGLO-Rab25-WT, pmirGLO-Rab25-MUT and a negative control using transfection reagent (R0531, Thermo, USA). At 48 h post-transfection, cells were lysed and luciferase activity was examined using the dual-luciferase reporter assay system (E1910, Promega, USA) according to the manufacturers’ instructions. All experiments were performed in triplicate and the results were expressed as firefly luciferase activity normalized to Renilla luciferase activity.

The second exon sequence of the INSR gene was inserted into the pCD2.1 vector (Geneseed Biotech, Guangzhou, China) and psi-CHECK2 vector (Promega, Fitchburg, WI, USA). Small interfering RNA (siRNA) oligonucleotides were designed to combine with the back-splice region of circINSR (RiboBio, Guangzhou, China). These siRNAs inhibited the expression of circINSR after transfection into the cell and was named si-circINSR. The mimics of bta-miR-15a, bta-miR-15b, bta-miR-16a, and bta-miR-16b were purchased from RiboBio (Guangzhou, China). The 3′-untranslated regions (UTRs) of the cyclin D1 (CCND1) and B-cell lymphoma 2 (Bcl-2) genes containing the miR-15/16 binding sites were amplified using the PCR enzyme mix (Platinum II Taq Hot-Start DNA Polymerase, Invitrogen). The wild-type and mutant 3'-UTR gene sequences were cloned into the psi-CHECK2 vector. The Renilla: Firefly ratio was measured and compared against that for the non-treated control. The mimics (50 nM) or vectors (2 μg/mL) were transfected into cells using a transfection reagent (R0531, Thermo Fisher Scientific, USA). For the overexpression of the miR-15/16 family, the miR-15a, miR-15b, miR-16a, and miR-16b mimics were mixed in equal amounts for transfection.