B04001

The binding sites between miR-23b-3p and NEU1 were predicted by TargetScan, and then dual-luciferase reporter assay was employed for confirmation.
NEU1 3′-UTR containing miR-23b-3p target sites obtained from Gene Pharma (Shanghai, China)was ligated into luciferase vector pMirGLO (AM5795, Thermo Fisher Scientific) to form reporter plasmids of wild-type NEU1 (NEU1-WT). QuikChange II Site-directed Mutagenesis Kit (200,523, Agilent, Santa Clara, CA) was used to perform 3′-UTR mutagenesis to create reporter plasmids of mutated NEU1 (NEU1-MUT). Then, for dual-luciferase reporter assay, we first cultured HEK-293 T cells in 96-well plate at an adjusted density of 5 × 10³ cells/well, and subsequently transfected 200 ng of miR-23b-3p mimic (M, sequence: 5′-AUCACAUUGCCAGGGAUUACCAC-3′; B02003, Gene Pharma, China), its control (MC, sequence: 5′-AUCAUAGGUCUCAUGGCCAACAC-3′; B04001, Gene Pharma, China), and 50 ng recombinant reporter plasmids of NEU1-WT (sequence: 5′-CUGUAGAAUUGAAUCAAUGUGAA-3′) and NEU1-MUT (sequence: 5′-CUGUAGAAUUGAAUCCGACCAUA-3′) into the cells using Lipofectamine 3000 reagent (L3000–001, Invitrogen). Following 48 h, HEK-293 T cells were harvested, and its luciferase activity was measured with dual-luciferase reporter assay system (E1910; Promega, Madison, MI). Renilla luciferase activity was used for firefly luciferase activity normalization.

HUVECs (Allcells, Shanghai, China) were cultured in HUVEC medium (HUVEC-004; Allcells) as previously described (Zeng et al., 2019 (link)). Cells (passages three to six) were maintained at 37°C with 5% CO₂ in a humidified incubator. Lentivirus infections of miR-9 mimic (5′-TCTTTGGTTATCTAGCTGTATGA-3′) and negative control (NC; 5′-UUGUACUACACAAAAGUACUG-3′; B04001; GenePharma, Shanghai, China) were performed as previously described (Zeng et al., 2019 (link)). cDNAs for S1P₁ were ligated into plasmid pcDNA3.1. For overexpression of S1P₁, cells were transfected with pcDNA3.1 vector expressing S1P₁ using Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, United States). Transfected cells after 48 h were used for the subsequent experiments. Recombinant human VEGF 165 Protein (50 ng/mL; hVEGF; 293-VE, R&D system, Minneapolis, MN, United States) was used to test the migration, invasion and in vitro angiogenesis of cells overexpressing S1P₁.