Pgl3 dual luciferase reporter vector

According to predictions made using the TargetScan database (release 7.2; http://www.targetscan.org), a putative miR-338-3p-binding site was identified in the 3′-untranslated region (3′-UTR) of the HOXA3 gene. The HOXA3 wild-type (WT) 3′-UTR sequence and a mutant version (MT) were amplified via PCR and cloned into the pGL3 dual-luciferase reporter vector (Promega Corporation) to create pGL3-HOXA3-3′-UTR-WT (WT vector) and pGL3-HOXA3-3′-UTR-MT (MT vector). Source of the DNA was human HOXA3 cDNA; polymerase: Pyrobest DNA polymerase (Fermentas; Thermo Fisher Scientific, Inc.). The primers used were: WT, forward, 5′-CCGCTCGAGGAGCCAGGAGTCACTAGG-3′, and reverse, 5′-TAAGCGGCCGCTTGTTTTCAGAACGTGAG-3′. The PCR settings were 94°C for 3 min; 35 cycles at 94°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec; terminated at 72°C for 5 min. SUP-T1 cells in the logarithmic growth phase were seeded into 96-well plates at 1.5×10⁴ cells/well, and co-transfected with the 100 ng WT or 100 ng MT vector and one of the 50 nM miRNA or 150 nM inhibitor (miR-338-3p mimics, NC, miR-338-3p inhibitor or inhibitor NC) using Attractene Transfection Reagent (Qiagen, Inc.). Following incubation for 48 h, the ratio of firefly to Renilla luciferase activity was determined using a dual-luciferase reporter system, according to the manufacturer's protocols (Promega Corporation).

Firstly, online software has validated the putative binding sites between miR-498 and AFAP1-AS1 or VEGFA using LncBase Predicted v.2.0 (http://carolina.imis.athena-innovation.gr/) and starBase v2.0 (http://starbase.sysu.edu.cn/mirCircRNA.php). In order to further confirm the relationships among them, we used Dual-luciferase reporter assay. Wild type AFAP1-AS1 fragments (AFAP1-AS1 WT) containing miR-498 binding sites or AFAP1-AS1 mutant sequence (AFAP1-AS1 MUT) without miR-498 binding sites was inserted into the pGL3 dual-luciferase reporter vector (Promega, Madison, WI, USA). After that, the dual-luciferase reporter plasmid of AFAP1-AS1 WT or AFAP1-AS1 MUT was co-transfected into Eca109 and KYSE-30 cells with NC mimics or miR-498 mimic by using Lipofectamine 3000 (Invitrogen). After transfection for 24 hrs, the relative luciferase activity was measured with the use of a dual-luciferase reporter assay system (Promega, Madison, WI, USA). Similarly, wild or mutant VEGFA 3ʹUTR sequence containing miR-498 binding sites or not was cloned into the pGL3 dual-luciferase reporter vector to form the reporter vectors VEGFA WT and VEGFA MUT.