Pgl 3.1 luciferase reporter vector

TargetScan (http://www.targetscan.org), a website widely used for prediction of microRNA targets, was applied to predict the targets of miR-22-3p. Based on the results, numbers of genes were the potential targets of miR-22-3p, including ENO1, SNAIL1, and RAC1. We focused on RAC1 gene in subsequent experiments. As shown in the website, the combination point of miR‐22-3p and RAC1 3′-UTR regions is GCAGCUC and GCAGCUU. The mutation sequence of RAC1‐3′-UTR is CGUCGAC and CGTCGAA, respectively. The 3′-UTR region (WT and Mut) of the RAC1 gene were sub-cloned into pGL 3.1 luciferase reporter vector (Promega). We cultured transfected 293 T cells into 96-well plates at a density of 5 × 10⁴ cells per well, and measured luciferase activity with the Dual-Luciferase Reporter Assay System kit (Promega) on EnSpire Multimode Plate Reader (PerkinElmer, Waltham, MA, USA) according to manufacturer’s instruction, with Renilla luciferase activity as normalized control.

The sequence 1 kb up-stream of the HPF1 gene and containing the HPF promoter was synthesized by Tianyihuiyuan (Beijing, China) and sub-cloned into the pGL 3.1 luciferase reporter vector (Promega). RKO cells were co-transfected with pGL 3.1-HPF1, pRL-TK vector, ANP32B siRNA or ANP32B overexpressing construct (pcDNA-ANP32B) using lipofectamine 2000. After 24 h, the cells were harvested and analyzed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions [24 ].