Pmirglo dual luciferase mirna target reporter vector

The target sequence of ANRIL was predicted through bioinformatics analysis. Wild type ANRIL (ANRIL-WT) or mutant ANRIL (ANRIL-MT) sequence was inserted into pmirGLO dual-luciferase miRNA target reporter vector (Promega, Madison, WI, USA). MOLT4 cells, CCRF-CEM cells and KOPT-K1 cells were inoculated in 24-well plates (5000 cells per well), and cultured for 24 h. After that, ANRIL-WT or ANRIL-MT reporter was co-transfected into the cells with miR-7-5p mimics or control microRNA, respectively. Then the cell culture was continued for 48 h, and the luciferase activity of each group was determined using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).

The targeted relationship between miR-625-5p and LINC00511 or the 3ʹ-untranslated region (3ʹ-UTR) of STAT3 was verified by a luciferase reporter gene assay. First, the wild type (WT) LINC00511 sequence or WT 3ʹ-UTR fragments from STAT3 mRNA containing predicted miR-625-5p binding sites were inserted into pmiRGLO dual-luciferase miRNA target reporter vector (Promega, Madison, WI, USA) to construct the report vector pmiRGLO-LINC00511-WT or pmiRGLO-STAT3-WT. Second, the putative binding site of miR-625-5p in the LINC00511 or STAT3 3ʹ-UTR was mutated by GeneArt™ Site-Directed Mutagenesis PLUS System (Thermo Fisher Scientific, Inc., Carlsbad, CA, USA). Third, the mutant (MUT) LINC00511 sequence or STAT3 3ʹ-UTR sequence was inserted into the pmiRGLO vector to construct the reporter vector pmiRGLO-LINC00511-MUT or pmiRGLO-STAT3-MUT. Fourth, the corresponding reporter vectors and miR-625-5p mimic or NC mimic were co-transfected into MKN28 cells and incubated for 48 h, and after that, the luciferase activity was subsequently evaluated by a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).