Pmirglo luciferase vector

EGFR 3'UTR region containing potential target sequences complementary to the miR-3622b seed sequence were cloned downstream of the luciferase gene in the pmiRGLO luciferase vector (Promega) according to the manufacturer's instructions. Mutated 3'UTR sequences complementary to miR-3622b were cloned in the same vector. Primers used for these clonings were synthesized by Invitrogen and are listed in Supplementary Table S1. 3'-UTR EGFR reporter/ control constructs (0.2 ug each) in the pmiRGLO luciferase vector (Promega) were each cotransfected in PC3 cells with 50 nM miR-CON/ miR-3622b precursor (Ambion) using Lipofectamine 2000 (Invitrogen). Firefly and Renilla luciferase activities were measured by using the dual luciferase reporter assay system (Promega) 48 hr post-transfection in accordance with the manufacturer's protocol. Firefly luciferase was normalized to Renilla luciferase activity.

The presumed binding site of miR-877-3p in NORAD and CREBBP was predicted on the LncBase Predicted v.2 and TarBase v.8 online tools (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php). The NORAD-3′-UTR fragments containing miR-877-3p wild-type (NORAD-WT) and mutant (NORAD-MUT) binding sites were inserted into the pmirGLO luciferase vectors (E1330, Promega, Madison, WI, USA). Additionally, the CREBBP-3′-UTR fragments containing miR-877-3p wild-type (CREBBP-WT) and mutant (CREBBP-MUT) binding sites were inserted into the pmirGLO luciferase vectors. Lipofectamine 3000 was used for cotransfection of NORAD-WT/NORAD-MUT and CREBBP-WT/CREBBP-MUT with miR-877-3p mimic or mimic NC and cultured for 48 h. Next, cells were harvested for the dual-luciferase assay (Promega). The luciferase activity was measured using the SpectraMax L fluorometer (Molecular Devices, Sunnyvale, CA, USA).

The wild-type or mutant circ_002136 sequence containing the miR-19a-3p binding site, the 3’-UTR wild-type or mutant sequence of RAB1A mRNA were inserted into the p-mir-GLO luciferase vectors (Promega, Madison, WI, USA) to obtain the desired luciferase reporter vectors (WT-circ_002136, MUT-circ_002136, WT-RAB1A and MUT-RAB1A). HCC cells were cotransfected with the above luciferase reporter vectors in 96-well plates, together with miR-19a-3p mimics or miR-NC. 48 h later, dual luciferase reporter gene assay system (Promega, USA) was constructed to quantify the corresponding luciferase activity.

The wild-type NEAT1 (NEAT1-WT), mutant NEAT1 (NEAT1-MUT), wild-type CTBP2-3′UTR (CTBP2-WT), and mutant CTBP2-3′UTR (CTBP2- MUT) were synthesized and cloned into pMIR-GLO™ Luciferase vectors (Promega, Madison, WI, USA). Luciferase reporter constructs containing the wild-type or mutated miR-129 (pMIR-miR-129-WT and pMIR-miR-129-MUT) were purchased from GeneChem (Shanghai, China). For the luciferase reporter assay, EC109 and EC9706 cells were cotransfected with constructed luciferase reporter vectors containing NEAT1 (WT or MUT) or CTBP2-3′UTR (WT or MUT) and miR-129 or miR-control. To investigate the effects of NEAT1 on wild-type or mutated miR-129, EC109 and EC9706 cells were cotransfected with miR-129-WT or miR-129-MUT reporter and pcDNA-NEAT1 or pcDNA. To determine the relationship between NEAT1, miR-129, and CTBP2, EC109 and EC9706 cells were transfected with CTBP2-WT, CTBP2-WT + miR-129, or along with pcDNA-NEAT1 or pcDNA. Lipofectamine 2000 reagent (Invitrogen) was utilized for all cell transfections. At 48 h posttransfection, cells were collected and assayed with the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to that of Renilla luciferase.