Pmirglo dual luciferase vector

To evaluate the mmu_circ_0005019 and miR-499-5p interaction by luciferase reporter assay, the miR-499-5p binding sites of mmu_circ_0005019 were inserted into the pmirGLO Dual-Luciferase vector (GenePharma). The reconstituted plasmid was named mmu_circ_0005019-WT. The miR-499-5p target site mutations were introduced and inserted into the pmirGLO Dual-Luciferase vector (GenePharma), which was named mmu_circ_0005019-MU. The plasmid construction was performed by GenePharma Corporation. HL-1 cells were seeded into 24-well plates (2 × 10⁴ cells per well) in triplicate for each group. After overnight incubation, cells were co-transfected with reconstituted plasmid and miR-499-5p mimics. Firefly and Renilla luciferase activities were measured 48 h after transfection using the Dual-Luciferase Assay System (Promega, Madison, WI, USA). The relative luciferase activity was calculated using Firefly/Renilla luciferase activity.

Wild-type and mutant KLF4 (with mutated miR-145 binding sites) were cloned into a pmirGLO dual luciferase vector (GenePharma) to construct dual luciferase reporter plasmids. HEK293T cells were cotransfected with wild-type pmirGLO-KLF4 (or mutant) and miR-145 mimics (or a negative control) using Lipofectamine 2000. Similarly, dual luciferase reporter plasmids containing TGFBR2-WT and TGFBR2-Mut were constructed. HEK293T cells were cotransfected with either miR-145 mimics/inhibitor or their corresponding empty vectors and luciferase reporter plasmids using Lipofectamine 2000. Luciferase activity was analyzed using a dual luciferase reporter kit (Promega, USA) after 48 h of transfection. Renilla luciferase activity was measured as a transfection efficiency control.

Potential miR-103a-3p binding sites of linc00152 and FEZF1 3′-UTR were predicted by bioinformatics tool Starbase (http://starbase.sysu.edu.cn/) and Target Scan (http://www.targetscan.org/). In brief, the fragment of linc00152 possessing the assumptive miR-103a-3p binding sites was cloned into a pmirGLO Dual-Luciferase Vector (Promega, Madison, WI, USA) to construct the reporter vector linc00152-wild-type (linc00152-Wt) (GenePharma, Shanghai, China). Analogously, the corresponding mutant of hypothetic miR-103a-3p binding sites was manufactured to form the reporter vector linc00152-mutated-types (linc00152-Mut) (GenePharma, Shanghai, China). The 3′-UTR fragment of FEZF1 gene and its mutant of the theoretical miR-103a-3p binding site were cloned into a pmirGLO Dual-Luciferase Vector to form the reporter vector FEZF1-3′-UTR-wild-type (FEZF1-3′-UTR-Wt) and FEZF1-3′-UTR-mutated-type (FEZF1-3′-UTR-Mut) (GenePharma, Shanghai,China), respectively. The pmirGLO vector(wild type fragments or mutated type fragments) and indicated miRNAs were transfected into HEK 293 T cells using Lipofectamine 3000. Relative luciferase activities were measured 48 h after transfection and firefly luciferase activity was normalized by renilla luciferase activity.