Nonspecific non coding sirna

By searching the Target Scan 6.2 database (http://www.targetscan.org/) to find algorithm-based binding sites of miR-145, we found a predicted binding site to be at positions 278–284 in the 3′-UTR of KLF4 mRNA. The sequence region 2161–2520, containing the putative binding sequence of miR-145, was inserted into a pMIR-REPORT^TM Luciferase miRNA Expression Reporter Vector (Applied Biosystems) according the manufacturer's protocol. Moreover, we made another pMIR construct encompassing a mutated seed sequence for miR-145 (Wild type, GACTGGAA; mutant, GACGTCAA) by using a PrimeSTAR^® Mutagenesis Basal Kit (TaKaRa). The mutation of the vector was confirmed by sequence analysis. The pRL-TK Renilla Luciferase Reporter vector (Promega, Madison, WI, USA) was used as an internal control vector. 253J B-V cells were seeded into 96-well plates at a concentration of 0.1 × 10⁴ per well on the day before the transfection. The cells were co-transfected with either reporter vector (0.01 μg/well each) and 20 nM miR-145 or nonspecific non-coding siRNA (Dharmacon, Tokyo, Japan). Luciferase activities were measured at 24 h after co-transfection by using a Dual-Glo Luciferase Assay System (Promega) according to the manufacturer's protocol. Luciferase activities were reported as the firefly luciferase/Renilla luciferase ratio.

We constructed a pMIR vector by inserting the sequence of a possible binding site in the 3′UTR of human DDX6 (No.3314-3320) into a pMIR-REPORT™ Luciferase miRNA Expression Reporter Vector (Applied Biosystems) according to the manufacturer’s protocol. Moreover, by using a PrimeSTAR^® Mutagenesis Basal Kit (TaKaRa), we also made another pMIR construct having a mutated seed sequence for miR-143 (wild type, CAUCUCA; mutant, CAAGACA). The sequence of this mutation vector was submitted to the Life Science Research Center, Gifu University for DNA sequencing. MKN-7 cells were seeded into 96-well plates at a concentration of 0.1 × 10⁴ per well on the day before the transfection. The cells were co-transfected with either reporter vector (0.01 μg/well each) and 20 nM syn-miR-143 or nonspecific non-coding siRNA (Dharmacon, Tokyo, Japan). Luciferase activities were determined by using the Dual-Glo Luciferase Assay System (Promega) and a GLOMAX 20/20 LUMINOMETER (Promega) at 24 h after co-transfection. The firefly luciferase activity was normalized by the co-transfected Renilla luciferase activity for determining co-transfection efficiency.

To identify the miR-122-5p binding sites, we used the Target Scan 7.2 database (http://www.targetscan.org/, accessed on 31 March, 2022) and found the predicted binding site to be at position 520–527 within the 3′UTR of PKM mRNA. The sequence region containing the putative binding sequence was inserted into a pMIR-REPORT^TM Luciferase miRNA Expression Reporter Vector (Applied Biosystems Inc.) according to the manufacturer’s protocol. We also generated other pMIR constructs, including a mutated seed sequence for miR-122-5p (wild-type, ACACUCC; mutant, ACUGACC), using a PrimeSTAR^® Mutagenesis Basal Kit (Takara Bio Inc.) [23 (link)]. The mutation of each vector was confirmed via sequence analysis. A pRL-TK Renilla Luciferase Reporter vector (Promega Corporation, Madison, WI, USA) was used as an internal control vector. Cells were seeded into 96-well plates at a concentration of 0.1 × 10⁴ per well on the day before transfection. Each cell type was co-transfected with a reporter vector (wild-type or mutant; 0.01 µg/well) and miR-122-5p or a non-specific non-coding siRNA (Dharmacon, Tokyo, Japan). Luciferase activity was measured using a Dual-Glo Luciferase Assay System (Promega Corporation) and reported as the Firefly luciferase/Renilla luciferase ratio [23 (link)].