Luciferase reporter vector

To confirm that DLEU1 and PRPS1 were targets of miR-320b, LncRNA DLEU1-WT (containing the binding sites of miR-320b at DLEU1), LncRNA DLEU1-Mut (mutation of binding sites), PRPS1-WT (containing the binding sites of miR-320b at PRPS1 3’UTR) and PRPS1-MUT (mutation of binding sites) were cloned into Luciferase Reporter Vector (Invitrogen, CA, USA). LncRNA DLEU1-WT, LncRNA DLEU1-Mut, PFKL-WT or PFKL-MUT were co-transfected with miR-320b mimics into LoVo and SW480 cells. After 48-h transfection, LoVo and SW480 were explored using a Reporter Vector System (Invitrogen, CA, USA) using a Glomax20/20 luminometer (Promega, WI, USA) (27 (link)).

HEK-293 cells were plated at a density of 4 × 10³ cells/well in 96-well plates 24 h before transfection. The cells were co-transfected with either anti-miR-320a (5′-UUUUCGACCCAACUCUCCCGCU-3′) or pre-miR-320a (5′-GCUUCGCUCCCCUCCGCCUUCUCUUCCCGGUUCUUCCCGGAGUCGG GAAAAGCUGGGUUGAGAGGGCGAAAAAGGAUGAGGU-3′, RiboBio, Guangzhou, China) at a final concentration of 50 nM using Lipofectamine 2000 (Invitrogen) followed by 100 ng/well Luciferase reporter vector containing the wild 3′-UTR of AQP₁ (forward primer, 5′-ATTAACTAGTCATTCCCTAGCA-3′ and reverse primer, 5′-TATGAAGCTTCAGGCAGG GGGT-3′) or mutant 3′UTR (underlined) AQP₁ (5′-CTGATTCCTCTCATTTAATTTGGCT-3′). Luciferase assays were performed with dual luciferase reporter assay system (Promega, Madison, WI) 48 h after transfection according to the manufacturer’s instructions. Renilla luciferase activity was normalized to firefly luciferase activity.

PFKL-WT (containing the binding sites of miR-185-3p at PFKL 3′-UTR) and PFKL-MUT (mutation of binding sites) were cloned into Luciferase Reporter Vector (AM5795, Invitrogen, CA, United States). PFKL-WT or PFKL-MUT was co-transfected with miR-185-3p mimic into H1299/ER or A549/ER cells. After transfection for 48 h, H1299/ER and A549/ER cells were evaluated using the Reporter Vector System (AM5795, Invitrogen, CA, United States) using a GloMax 20/20 Luminometer (Promega, WI, United States) (Zhang et al., 2020 ).