The Firefly luciferase-UTR reporter plasmid was constructed by introducing the 3′-UTR of VEGFA (NM_ 001025366.2), FAK (PTK2) (NM_005607.4), ANGPT2 (NM_001147.2), and HIF1A (NM_001530.3) genes into pmiR-REPORT miRNA Expression Reporter Vector System (Life Technology). The 3′-UTR sequence of the four analysed genes was amplified by PCR from HEK 293 cDNA. For ANGPT2 two different constructs were made, pMIR-ANGPT2-573 containing the hypothetical seed for hsa-miR-573 and pMIR-ANGPT2-578 for hsa-miR-578, respectively. All constructs were verified by sequencing.
The reporter construct (50 ng), pSV-Renilla (2 ng, pRNL-SV40, Promega), and hsa-miR-573 or hsa-miR-578 mimic, or miRNA negative control (Sigma) were transfected into HEK293 cells using Lipofectamine 2000 (Life Technologies). Two different amount of miRNA mimic (1 and 2 pmoles) were used. After 48 hours, the cells were lysed in passive lysis buffer and assayed for both firefly and renilla luciferase activity using the Dual-GLO® Luciferase Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity for each transfected well. Values are the mean ± S.E.M. of three experimental replicates from three independent transfections. Significance was determined by a two-tailed unpaired t test for means.
The reporter construct (50 ng), pSV-Renilla (2 ng, pRNL-SV40, Promega), and hsa-miR-573 or hsa-miR-578 mimic, or miRNA negative control (Sigma) were transfected into HEK293 cells using Lipofectamine 2000 (Life Technologies). Two different amount of miRNA mimic (1 and 2 pmoles) were used. After 48 hours, the cells were lysed in passive lysis buffer and assayed for both firefly and renilla luciferase activity using the Dual-GLO® Luciferase Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity for each transfected well. Values are the mean ± S.E.M. of three experimental replicates from three independent transfections. Significance was determined by a two-tailed unpaired t test for means.