Pmir report reporter

The binding sites between miR-615-5p and circ-OGDH or PDX1 were predicted using circular RNA interactome and TargetScan. The fragments of wild type (wt) circ-OGDH and 3ʹ untranslated regions (UTR) of PDX1 and their mutant (mut) sequences were inserted into the downstream of the pMIR-REPORT reporter (Applied Biosystems), respectively. ESCC cells were co-transfected with a luciferase reporter containing wt-circ-OGDH, mut-circ-OGDH, wt-PDX1 3ʹUTR, or mut-PDX1 3ʹUTR and miR-NC or miR-615-5p mimic. Thereafter, the cells were lysed and the luciferase activities were assessed with a luciferase assay kit (Biovision) in a TD20/20 Luminometer (Turner Biosystems, Sunnyvale, CA, USA).

In order to verify the putative binding sites, the luciferase reporters containing the sequence of circ_0001944-wt (wild type), circ_0001944-mut (mutant), NFAT5 3ʹUTR (untranslated region)-wt, and NFAT5 3ʹUTR-mut were constructed using the pMIR-REPORT reporter (Applied Biosystems, Foster City, CA, USA), respectively. NSCLC cells were transfected with miR-142-5p mimic or miR-NC together with a luciferase reporter carrying circ_0001944-wt, circ_0001944-mut, NFAT5 3ʹUTR-wt, or NFAT5 3ʹUTR-mut, followed by evaluating the luciferase activity using a dual-luciferase reporter assay kit (BioVision, Milpitas, CA, USA).

MiRs that might interact with circATXN7 were jointly predicted using circBank, starBase, and CircInteractome databases. The binding sites between miR‐7‐5p and PFN2 3′ untranslated region (UTR) were predicted using the TargetScan database. To verify the relationship between circATXN7 or PFN2 and miR‐7‐5p, the wild‐type (WT) sequences of circATXN7 and PFN2 3′UTR and their mutant (MUT) sequences were inserted into the pMIR‐REPORT reporter (Applied Biosystems), respectively. NSCLC cells were transfected with miR‐7‐5p mimic or miR‐NC together with a luciferase reporter. The luciferase activity was assessed using the Pierce Renilla‐Firefly Luciferase Dual Assay Kit (Invitrogen).