Pmir rb report dual luciferase reporter gene plasmid vector

For confirmation of direct target binding, the wild type (smad2 WT) and mutant (smad2 MUT) 3′UTR of smad2 identified by TargetScan were cloned into a pmiR-RB-ReportTM dual luciferase Reporter gene plasmid vector (Guangzhou RiboBio Co., Ltd.). The UTR region of candidate target gene was inserted downstream of the sequence of Renilla luciferase, which was designed for reporter fluorescence (Rluc). For luciferase reporter analysis, luciferase reporter vectors and mimic control, miR-205-5p mimics, were transfected into HSF cells using Lipofectamine 2000. After 48 h, luciferase activity was analyzed by the Dual-Luciferase Assay System (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocols.

In the current study, TargetScan bioinformatics software version 7.1 (www.targetscan.org/vert_71) was used to predict the targets of miR-15a-5p, and it was revealed that TNIP2 was a potential target of miR-15a-5p. To confirm the binding sites between miR-15a-5p and TNIP2, the wild type (WT-TNIP2) and mutant (MUT-TNIP2) 3'UTR of TNIP2 were cloned into a pmiR-RB-ReportTM dual luciferase reporter gene plasmid vector (Guangzhou RiboBio Co., Ltd). Subsequently, 293T cells were cotransfected with WT-TNIP2 or MUT-TNIP2 and miR-15a-5p mimic or mimic control using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), as per the manufacturer's protocols. After 48 h of cell transfection, luciferase activity was determined using the dualluciferase assay system (Promega Corporation) in line with the manufacturer's protocol. Luciferase activity was normalized to Renilla luciferase activity.

Next, the mechanism by which miR-221 acted on human T-ALL cell lines was investigated. The target genes of miR-221 were searched using miRanda (microRNA.org), and PTEN was found to be a potential target of miR-221. To confirm the binding sites between miR-221 and the 3'-UTR of PTEN, a dual-luciferase reporter assay was performed. The wild-type (WT-PTEN) and mutant (MUT-PTEN) 3'-UTRs of PTEN were cloned into a pmiR-RB-Report™ dual-luciferase reporter gene plasmid vector (Guangzhou RiboBio Co., Ltd.). The MUT 3'-UTR of PTEN was constructed using a QuikChange Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc.) according to the manufacturer's instructions. Jurkat cells (5x10⁴ cells/well) were transfected with the reporter constructs and miR-221 mimic (5'-AGCUACAUUGUCUGCUGGGUUUC-3'; Guangzhou Ribobio Co., Ltd.) or mimic control (5'-CGGUACGAUCGCGGCGGGAUAUC-3'; Guangzhou Ribobio Co., Ltd.) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Luminescence was assayed 48 h later using the Dual-Luciferase Reporter Assay System (Promega Corporation) according to the manufacturer's instructions. Results were normalized to the Renilla luminescence from the same vector and shown as the ratio between the various treatments and cells transfected with control vector.

Bioinformatics software (TargetScan 7.2, http://www.targetscan.org/vert_72/) was used to predict target gene of miR-217. The results revealed the binding sites between the 3′-UTR of SIRT1 and miR-217. To verify whether miR-217 could target SIRT1, we performed a dual-luciferase reporter assay. The wild type (WT-SIRT1) and mutant (MUT-SIRT1) 3′-UTR of SIRT1 were respectively cloned into a pmiR-RB-Report™ dual luciferase reporter gene plasmid vector (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions. To point-mutate the miR-217 binding domain on the 3′UTR of SIRT1, a QuikChange Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc.) was performed following the manufacturer's instructions. Then, 50 nM miR-217 mimic (agomir; 5′-UACUGCAUCAGGAACUGAUUGGA-3′; Shanghai GenePharma Co., Ltd.) or 50 nM mimic control (5′-UUUGUACUACACAAAAGUACUG-3′; Shanghai GenePharma Co., Ltd.), and the WT or MUT 3′-UTR of SIRT1 were co-transfected into ARPE-19 cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h. Subsequently, relative luciferase activity was detected using a Dual Luciferase Assay System (Promega Corporation) with a microplate reader (Molecular Devices, LLC). Renilla luciferase was used for the normalization of firefly luciferase activity. Each experiment was performed three times.

A bioinformatics prediction program (TargetScan 7.2; http://www.targetscan.org/vert_72/) was used to predict the relationship between miR-24-3p and Bcl-2L11, and binding sites between miR-24-3p and Bcl-2L11 were observed. To confirm the prediction, the wild-type (WT-Bcl-2L11: 5'-CCCCUGCAGUGGAAACUGAGCCA-3') and mutant (MUT-Bcl-2L11: 5'-CCAAGCAAGUGGAAAAGCGCAAG-3') 3'UTR of Bcl-2L11, containing the miR-24-3p-binding elements, were generated by RT-PCR using a Transcriptor First Strand cDNA Synthesis Kit (Roche Molecular Systems, Inc.) from total RNA preps extracted from VSMCs, using the temperature protocol of 5 min at 25˚C followed by 60 min at 42˚C. The sequences were then cloned into a pmiR-RB-Report™ dual luciferase reporter gene plasmid vector (Guangzhou RiboBio Co., Ltd.). Then 100 ng Bcl-2L11-WT or 100 ng Bcl-2L11-MUT were co-transfected with 100 nM miR-24-3p mimic or 100 nM mimic control into VSMCs using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. Dual-Luciferase^® Reporter Assay kit (Promega Corporation) was used to measure luciferase activity 48 h after cell transfection, which were normalized to that of Renilla luciferase.

TargetScan version 7.2 (http://www.targetscan.org/vert_72/) was used to predict the potential targets of miR-133a-3p, and the binding sites between miR-133a and MMP-9. To confirm the association between miR-133a-3p and MMP-9, a dual-luciferase reporter assay was performed.
The wild-type and mutant 3′-UTR of MMP-9 (WT-MMP-9 and MUT-MMP-9, respectively) were cloned into a pmiR-RB-Report™ dual luciferase reporter gene plasmid vector (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's protocol. hVSMCs were first seeded into 24-well plates (5×10⁴ cells per well) and then co-transfected with 100 ng WT-MMP-9 or 100 ng MUT-MMP-9 and 100 nM miR-133a-3p mimic or 100 nM mimic control using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol, together with Renilla luciferase pRL-TK vector (Promega Corporation) as a control. Following transfection for 48 h, the relative luciferase activity was measured using the dual-luciferase reporter assay system (Promega Corporation), as per the manufacturer's protocol. All firefly luciferase activities were normalized to Renilla luciferase activity.

To predict the targets of miR-18a-5p, TargetScan bioinformatics software (www.targetscan.org/vert_71) was used, and results revealed that Smad2 was a potential target of miR-18a-5p. To confirm the direct binding sites, the wild-type (WT-Smad2) and mutant (MUT-Smad2) 3′-untranslated region (3′-UTR) of Smad2 were cloned into a pmiR-RB-Report™ dual luciferase reporter gene plasmid vector (Guangzhou RiboBio Co., Ltd., Guangzhou, China). To point-mutate the miR-18a-5p binding domain on the 3′UTR of Smad2, a QuikChange Site-Directed Mutagenesis Kit (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA) was used as per the manufacturer's instructions. SCC9 cells were co-transfected with 100 ng WT-Smad2 or 100 ng MUT-Smad2 and 50 nM miR-18a-5p (miR mimic) or 50 nM negative control (NC) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocols. Luciferase activity was assessed, 48 h later, using the Dual-luciferase assay system (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. Luciferase activity was normalized to Renilla luciferase activity.