4.12. Luciferase miRNA Target Reporter Assay

Targetscan 7.2 2020 (http://www.targetscan.org, accessed on 24 May 2021) was used for miRNA target prediction. Wild type (WT) and mutant (MUT) vectors of BCL2L1-3′UTR and BCL2-3′UTR were purchased from GeneCopoeia (Rockville, MD, USA). The full length BCL2L1-3’UTR (217HmiT108616-MT05; 1489 bp) and of the proximal part of BCL2-3’UTR (217HmiT016211a; 2761 bp) were inserted downstream of a Gaussia luciferase (GLUC) reporter gene in the pEZMX-MT05 vector. This vector also carries the secreted alkaline phosphatase (SEAP) reporter gene for normalization of GLUC luciferase activity, as a function of transfection efficiency. SW1353 cells were seeded at 8 × 10⁴ cells/well (triplicates) in a 24-well plate the day before transfection. The cells were co-transfected with 20 nM miRNA mimic or miRNA hairpin inhibitor and with BCL2-3′UTR vector (0.25 ng/µL) using jetOPTIMUS^TM DNA transfection reagent (Polyplus-Transfection, Strasbourg, France). BCL2L1-3′UTR vector (1 ng/µL) and miRNA were co-transfected with EndoFectin transfection reagent (Genecopoeia, Rockville, MD, USA). The culture medium was collected 24 h later to measure luciferase activities with the secrete-pair dual luminescence assay kit (Genecopoeia, Rockville, MD, USA) and a luminescence microplate reader (Spark 10M, Tecan, Lyon, France). Luciferase activities were expressed as relative luciferase units (RLU) corresponding to GLUC/SEAP ratio.