Site direct mutagenesis kit

TargetScan version 7.2 (targetscan.org/vert_72/) analysis was performed to determine the potential targets of miR-190b and a total of 223 genes were identified. Among these genes, FOXP2 was selected for following studies. Wild-type 3′-UTR of FOXP2 was amplified from the human CRC cell genome with the primers (Sense, 5′-GGTACCCTTTACAAACAGTTTTGACAG-3′, and antisense, 5′-AAGCTTTGGTGTGAATGATGACTGG-3′) and cloned into pGL3 luciferase vector (Promega Corporation) and termed pGL3-FOXP2-wt. A site-direct mutagenesis kit (Takara Bio, Inc.) was used mutate the FOXP2 3′-UTR using the primers (Fragment 1, forward 5′-CAGCGATCGCGAACTGACTTGTGAAACCTCAGCG-3′, reverse, 5′-CTCGCAGTTACTTCCAGTCCCTCAAAGCC-3′; and fragment 2, forward 5′-GTCTTTGGGTCATGATCAACGAACCGG-3′ and reverse, 5′-TATGTTTAAACTTTATAAATGGGTCAAAAAGAATTAGA-3′) and this was termed pGL3-FOXP2-mut. Cells were co-transfected with pGL3-FOXP2-mut or pGL3-FOXP2-wt combined with miR-190b inhibitor or NC-inhibitor using Lipofectamine^® 2000. Luciferase activity was measured using a Dual-Luciferase reporter system (Promega Corporation) after 48 h of transfection.

Wild-type (wt) sequence of CASC9 or S100A14 contains a binding region for miR-335-3p was inserted into psi-CHECK-2 (Promega. Madison, WI, USA) to generate wt-CASC9 or wt-S100A14. Mutant luciferase vectors (mt-CASC9 or mt-S100A14) were constructed using site-direct mutagenesis kit (Takara) to lose miR-335-3p binding sites. Cells were co-transfected with luciferase vectors and miRNAs using Lipofectamine 2000. 48 hrs later, dual-luciferase activity system (Promega) was utilized to measure relative luciferase activity using Renilla luciferase as internal control.

pmiR-Report luciferase vector contains wild-type (wt) sequence of PART1 or HMGB2 was named as wt-PART1/HMGB2. Mutant luciferase vectors were constructed using site-direct mutagenesis kit (Takara) and named as mt-PART1/HMGB2. Cells were transfected with luciferase reporter vectors and miRNAs using Lipofectamine 2000. After 48 h transfection, relative luciferase activity was measured using Dual-luciferase reporter assay kit (Promega, Madison, WI, USA).