Generate site directed mutagenesis system

The Fn1 3′-UTR was amplified by PCR using the primers 5′-ACGTTTGCTAGCATCTTTCCAGCCCCACCCTA-3′ and 5′-ACGTTT GTCGACgacaatttttCTCTCCAAACACA-3′ and cloned into the NheI and XhoI sites of pSGG vector. The plasmid obtained was then used as template to generate a mutant miR response element for miR-140-3p using the Generate site-directed mutagenesis system (Invitrogen, Carlsbad, CA) and primers 5′-tctttttattaaaacacttgtctttcgagactagtaaagcgttggcatgtgcttat-3′ and 5′-ataagcacatgccaacgctttactagtctcgaaagacaagtgttttaataaaaaga-3′. The mutant contained three point mutations, tacta(c to g)t(g to c)t(g to c)gaaagacaa, as confirmed by sequencing. HEK293T cells were seeded in twelve-well plates (3 × 10⁵/well) and transfected with pGL3 luciferase vector containing wild type fibronectin 3′-UTR or mutant fibronectin 3′-UTR by Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Cells were co-transfected with miR-140 overexpressing plasmid. Luciferase activity was determined using the Dual-Luciferase assay system (Promega, Madison, WI) after 48 h of transfection. Luciferase activity was normalized to Renilla luciferase activity.

A MT1JP fragment containing putative miR-24-3p target sites was amplified by PCR using 5’-CTCCTGCAAGAAGAGCTGC-3′ and 5’-TGCAGCAAATGGCTCAGTA-3′ primers, and cloned into the NaeI and HindIII sites of a pMIR-7 REPORT vector (Ambion). This reporter construct was named wild-type-MT1JP (WT-MT1JP). To generate a mutant reporter plasmid, we used a Generate site-directed mutagenesis system (Invitrogen) to introduce mutations into the putative miR-24-3p target sites of the wild-type-MT1JP vector. A mutant reporter (MUT-MT1JP) was constructed in which “GGCUCAGU” [71825078–718,250,786 nt] was converted into “CCGAGTCG”. The reporter plasmid was transfected into cells using Lipofectamine 2000. To correct for transfection efficiency, an empty luciferase reporter vector without the miR-24-3p target was transfected in parallel. Luciferase activities in cells were expressed as ratios of the luciferase activity of the reporter vector with the miR-24-3p targeting sequence over the one without the targeting sequence. After 48 h of transfection with or without miR-24-3p inhibitor, cells were harvested and luciferase activity was measured using a Dual-Luciferase Assay Kit (Promega, Madison, WI, USA).