Pmir rb report dual luciferase reporter vector

TargetScan bioinformatics software version 7.2 (www.targetscan.org/vert_72) was used to predict the potential targets of miR-320a. Binding sites between miR-320a and the 3'-untranslated region (3'-UTR) of TUSC3 were observed. Dual luciferase reporter assay was performed to determine whether miR-320a directly bound to TUSC3. Wild-type (WT) and mutant (MUT) 3'-UTR of TUSC3 were cloned into pmiR-RB-Report™ dual luciferase reporter vector (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions. 293 T cells (American Type Culture Collection) were co-transfected with WT-TUSC3 or MUT-TUSC3 and 100 nM miR-320a mimic (5'-AAAAGCUGGGUUGAGAGGGCGA-3'; 3'-UUUUCGACCCAACUCUCCCGCU-5'; Guangzhou RiboBio Co., Ltd.) or 100 nM mimic control (5'-UUCUCCGAACGUGUCACGUTT-3'; 3'-TTAAGAGGCUUGCACAGUGCA-5'; Guangzhou RiboBio Co., Ltd.) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C for 48 h. At 48 h after transfection, luciferase activity was determined using the Dual-luciferase^® Reporter Assay system (Promega Corporation) and normalized to Renilla luciferase activity.

The bioinformatics software predicted the binding between miR-495 and GRP78 mRNA. RT-PCR was used to amplify a sequence encompassing these 501 base pairs. The primers for amplification were designed as follows: GRP78_WT_forward, 5′-ACTGCTGTTTTCAGATGGAGGT-3′ and reverse, 5′-CTAGGAGCCAGCTCAGATGC-3′; GRP78_mut_forward, 5′-TGCGGAGATCTATCTATCATGGC-3′ and reverse, 5′-GGTGTCAGGCGATTCTGGTC-3′. The amplified fragments were cloned into the pmiR-RB-REPORT™ dual luciferase reporter vector (Guangzhou RiboBio Co., Ltd., Guangzhou, China). The hRluc vector was used to report fluorescence, and the 3′UTR of GRP78 was cloned downstream of the hRluc gene. The directly targeted region was determined by cloning the 3′UTR seed region and the mutated seed region into the pmiR-RB-REPORT™ luciferase reporter vectors (Guangzhou RiboBio Co., Ltd.).