Pmir control vector

Using TargetScan,¹SLUG and SMAD3 were identified as miR-203 targets. The 3′-UTR of canine SLUG or SMAD3, which included the biding site of miR-203, was amplified using LA Taq (Takara, Shiga, Japan) with the following primers: SLUG, sense; 5′-CTG ACA GCT AGA TTG AGA GG, antisense; 5′-GCC AAT AAG GAG TAT GCA CC, and SMAD3, sense; 5′-AAT TGC AGG CTT GGT GCA GA, antisense; 5′-GCA GGT CTG TAT CTC AAA TG. A sensor vector was constructed by combining the amplified region with the luciferase reporter pMIR-control vector (Ambion, Foster City, CA, United States). In addition, to generate the sensor vectors with mutations in the binding site for miR-203, seed regions were mutated from CATTTCA to GCAATGC using a PrimeSTAR^® Mutagenesis Basal Kit (Takara, Otsu, Japan). The sequence of the mutated sensor vector was confirmed by Fasmac (Atsugi, Japan). The cells were seeded in 12-well plates at a concentration of 0.5 × 10⁵/well the day before transfection. The sensor vector at a concentration of 1.0 μg/well and 40 nM miR-203 or non-specific control miRNA were co-transfected into the cells using Lipofectamine RNAiMAX. At 48 h after co-transfection, the luciferase activity was measured using the Dual-Glo^™ Luciferase Assay System (Promega, Madison, WI, United States) following the manufacturer’s instruction. The luciferase activity of firefly was normalized to that of Renilla.

We constructed the sensor vector by joining the regions with a possible binding site from the 3′-UTR of canine PTEN to a luciferase reporter pMIR-control vector (Ambion, Foster City, CA, USA) to examine the target sequence recognized by miR-374b. Moreover, to generate the sensor vectors with mutations in the binding site for miR-374b, we mutated seed regions from TATTATA to GCAAGCT in region A and CGCATGT in region B (Figure 3A) by using a PrimeSTAR^® Mutagenesis Basal Kit (TaKaRa, Otsu, Japan). The sensor vector with mutations was submitted to Fasmac (Atsugi, Japan) for DNA sequencing. The cells were seeded in 12-well plates at a concentration of 0.5 × 10⁵/well the day before the transfection. The sensor vector (concentration; 1.0 μg/well) and 40 nM miR-374b-5p or non-specific control miRNA was used for the co-transfection of the cells by using cationic liposomes Lipofectamine RNAiMAX. Forty-eight hours after the co-transfection, luciferase activities were measured by using a Dual-Glo™ Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Firefly luciferase activity was normalized to Renilla luciferase activity.

We constructed sensor vectors by joining the region with or without a possible binding site from the 3′-UTR of human E2F3 (No. 4271-4650) with a luciferase reporter pMIR-control vector (Ambion, Foster City, CA, USA) to examine the target sequence of MIR34a. To generate sensor vectors with four mutations in the binding site of the 3′-UTR of human E2F3 (No. 4440-4462) for MIR34a, we mutated seed regions from CACTGCCA to CAAGTGCA (mt-E2F3, PrimeSTAR Mutagenesis Basal Kit; TaKaRa). The sensor vector with these mutations was submitted to Life Science Research Center, Gifu University, for DNA sequencing. The cells were seeded in 12-well plates at a concentration of 0.5 × 10⁵/well the day before transfection. The sensor vector (concentration, 0.5 μg/well) and 40 nM MIR34a or nonspecific control miRNA (Dharmacon) was used for the co-transfection of the cells with Lipofectamine RNAiMAX (Invitrogen). The measurement of the activities was described previously^{12 (link)}.