Pmir repor luciferase

Gene prediction software (https://cm.jefferson.edu/rna22/Precomputed/) was adopted to predict the target gene ofmiR-99a. The 3’UTR fragments of BRD4 containing both mutant (MUT) and wild-type (WT) binding sites of miR-99a were amplified by polymerase chain reation (PCR) and cloned into the vector pMIR-REPORLuciferase (Promega, Madison, WI, United States of America) to form luciferase reporter vectors. HEK-293T cells (Invitrogen Life Technologies, Carlsbad, CA, United States of America) were spread on 96-well plates at 4 × 103 (link) cells/well. HEK-293T cells were transfected withmiR-99a mimic + BRD4-wild type (WT), miR-99a mimic + BRD4-mutant type (MUT), mimic NC + BRD4-WT, or mimic NC + BRD4-MUT with lipofectamine 2000 (Invitrogen). At 48 h after transfection, cells were harvested, and luciferase activity was measured using dual luciferase reporter gene kit (Promega, Madison, WI, United States of America). The ratio of firefly luciferase activity to Renilla luciferase activity indicated the relative activity of luciferase21 (link).

The target gene statistics of miR-183 were carried out by using database TargetScan. IRS1 was preliminary selected as the direct target gene of miR-183. After inoculating cardiacmyocytes at the well plate, the total length of the 3′ UTR region of the wild-type (wt) IRS1 gene was cloned and amplified when the cell confluency reached about 70%. The PCR product was cloned into pMIR-REPOR™ Luciferase (Promega Corporation, Madison, WI) vector and named as pMI/IRS1-wt vector. The pMIR/IRS1-mutant type (mut) vector was constructed by site-directed mutation of the binding site between miR-183 and target gene, which was predicted by bioinformatics information. The vector was used to adjust cell number and transfection efficiency by using phRL-TK vector expressing Renilla luciferase (TaKaRa company) as internal reference. Two reporter gene vectors (pMIR/IRS1-wt and pMIR/IRS1-mut) and miR-183 mimic and their NC sequences (miR-183 NC) were co-transfected into cardiomyocytes to detect the activity of double luciferase according to the method provided by Promega Corporation (Madison, WI).

The 3′UTR fragments of PTEN containing both mutant (MUT) and wild-type (WT) binding sites of miR-26a-5p were amplified by PCR and cloned into the vector pMIR-REPOR™ Luciferase (Promega Corp., USA) to form luciferase reporter vectors. The vectors and miR-26a-5p mimic were co-transfected into primary cardiomyocytes via Lipofectamine 3000 (Invitrogen). Luciferase activity after 24 h of transfection was determined by a dual luciferase reporter assay kit (Promega) on a Fluoroskan Ascent Type 379 fluorescence plate reader (Thermo), and normalized to renilla luciferase intensity.