Hmit008690 mt06

The luciferase assay was used to assess whether miR-378a-5p targets the 3’ UTR of GZMB mRNA. A pEZX-MT06-GZMB-LUC reporter plasmid containing 113 bp of human GZMB 3’ UTR (HmiT008690-MT06) and negative control with no 3’ UTR (pEZX-MT06) was purchased from GeneCopoeia^™, Rockville, MD. HEK293T cells were plated 24 hours prior to transfection in 96-well plates (3×10⁴ cells/well). pEZX-MT06-GZMB-LUC or pEZX-MT06 plasmids were co-transfected with or without miR-378a-5p mimic or inhibitor using Attractene transfection reagent (Qiagen). Cells were harvested 24 hours after transfection. Firefly and renilla luciferase activities were determined using the Luc-Pair^™ Duo-Luciferase Assay kit 2.0 (GeneCopoeia^™, Rockville, MD). Relative luciferase activities were calculated by normalization of firefly luciferase activity to renilla luciferase activity to correct for transfection efficiencies in each sample and compared to cells transfected with the negative control plasmid (pEZX-MT06) or GZMB-Luc plasmid (pEZX-MT06-3’UTR GZMB-Luc).