Dual luciferase reporter detection system

We cloned wild‐type and mutant MiRNA‐96‐5p binding sequences from FOXO3 3′UTR into the pGL3 Basic vector (Promega, Madison, Wisconsin). MiRNA‐96‐5p and the 3′UTR (wild or mutant) of the FOXO3 recombinant plasmid were cotransfected into 293T cells. Transfection was performed using Lipofectamin 3000 reagent (Invitrogen), and luciferase activity was detected using a dual luciferase reporter detection system (Promega, Madison, Wisconsin).

The miRNAs interacted with PVT1 and target genes of miR-503 were predicted by online software miRcode and Target Scan Human 7.2, respectively. The wide-type luciferase reporters (WT-PVT1 and WT-ARL2) were generated by cloning fragments of PVT1 and ARL2 3′-UTR harboring binding sites (5′-GCUGCUA-3′) with miR-503 into pGL3 luciferase promoter vectors (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Likewise, mutant ones were established by inserting fragments containing the corresponding mutant-binding sites (5′-CGACGAU-3′). These reporters were severally cotransfected into CC HeLa and SIHA cells with pRL-TK Vector (Promega; an internal control) and miR-503 or miR-NC using Lipofectamine 3000 (Life Technologies). After 48 h, luciferase activity was measured using Dual-Luciferase Reporter detection System (Promega).

Previously, we obtained the pCMV-CRTC2-flag plasmid described and provided by Dr. Jeffery L. Meier (Infectious Disease Department, Carver School of Medicine, University of Iowa). The plasmid contains human CRTC2 that is C-terminally tagged with Myc and Flag, and we used lipofectamine 3000 reagent (Invitrogen) for transfection analysis according to the manufacturer's instructions. For reporter gene detection, cells were co-transfected with 0.5 mg E-cadherin or α-SMA luciferase with or without CRTC2-Flag plasmids, CREB siRNA, or Smad2/3 siRNA, and the internal reference was the renilla luciferase plasmid pRL-SV40 (Promega, Madison, WI). After 48 h, we washed once with PBS and shake the culture plate in 1 × buffer (Promega) to harvest the cells. According to the manufacturer’s protocol, the dual-luciferase reporter detection system (Promega) was used to measure the activity levels of firefly and kidney cell luciferase in the cell lysate. The level of firefly luciferase activity in the lysate of transfected cells was normalized to the level of renal cell luciferase activity.

To perform the dual-luciferase reporter gene assay, we designed a wild-type or mutant TIMP2 3’UTR luciferase reporter vector, the 3’UTR sequence of TIMP2 including the fragment with predicted binding site for miR-93 or mutant were cloned into the pGL3-promoter vector. The vector was co-transfected with miR-93 mimics/inhibitor or negative control using Lipofectamine 2000. PGL-TK vector expressing Renilla luciferase was served as the transfection control. After co-transfection for 48 h, Luciferase activities containing Renilla luciferase and firefly were evaluated using a Dual-Luciferase Reporter detection System (Promega, Madison, WI, USA) following the manufacturer’s instructions. For each assay, the luciferase activity was performed in triplicate.

A double luciferase reporter plasmid was constructed using Genescript (Nanjing, China), including pmiR-TUSC7-wt (containing miR-23b target binding sequence in TUSC7) and pmiR-MACC1-mut (containing mutational binding sequence). The dual luciferase assay is performed by co-transfection of pmiR-TUSC7-wt or pmiR-MACC1-mut and agomir-23b or agomir-NC into peritoneal macrophages. Luciferase activity after 48 h of co-transfection of peritoneal macrophages was detected with the Dual Luciferase Reporter Detection System (Promega, USA) in accordance with the manufacturer’s protocol.

HIPK1 3ʹuntranslated region (UTR) consisting of wild-type (WT) or mutant (MUT) binding sites of miR-30 c-5p was cloned into the pmirGLO vector (Promega), named HIPK1-WT and -MUT. HT22 cells were seeded into 24 well plates at a concentration of 2 × 10⁴ cells/well, and transfection of the cells was with HIPK1-WT and -MUT, and miR-30 c-5p mimic or its NC (RiboBio). Detection of the luciferase activity was via dual luciferase reporter detection system (Promega) [36 (link)].