Pmirglo dual luciferase plasmid

Dual-luciferase reporter assay was performed, as described previously (Wang et al., 2018 (link)). Briefly, after amplification through polymerase chain reaction, part of the DNA sequences of CDC25A or lncRNA SNHG11 with mutant (MU) or wild-type (WT) miR-184 binding sites were subsequently cloned to a pmirGLO dual-luciferase plasmid (Promega, Madison, WI) to prepare SNHG11-WT, SNHG11-MU, CDC25A-WT, and CDC25A-MU reporter plasmids that were individually transfected with miR-184 or NC mimics into HEK293T cells. When completed, the luciferase activities were assessed using the dual-luciferase reporter assay system (Promega).

The 3′ UTR regions of INSIG1, INSIG2, and FOXO1 were amplified using genomic DNA isolated from HepG2 cells and the primers FOXO1, 5′-TCTAGAGGGTTAGTGAGCAGGTTACACTTAA-3′ and 5′-GTCGACAGGTCCAAGGCTGTTCAATGGAGAT-3′; INSIG1A, 5′-TCTAGAAGATCGGGCTGACTGTACAAATGAC-3′ and 5′-GTCGACATTGTCTACACAAACTGCCACGGGA-3′; INSIG1B, 5′-TCTAGATCAGCAGAATGGAAGCTTAGAGGAA-3′ and 5′-GTCGACCTTAGTATGAATGTGAACCTCACTAG-3′; INSIG2, 5′-TCTAGATACTGCAATCTGTGATTGCTTCATC-3′ and 5′-GTCGACTCTGCTCATCACATATACTTCCAGT-3′. PCR products were digested using XbaI and SalI and inserted into the XbaI and SalI sites of a pmirGLO dual-luciferase plasmid (Promega). Resulting plasmids were designated pFOXO1, pINSIG1A, pINSIG1B, and pINSIG2. The putative binding sites of miR-183 in INSIG1A and miR-96 in INSIG2 were deleted from pINSIG1A and pINSIG2 plasmid using a Quick Change Lightning Multi Site Directed Mutagenesis Kit (Agilent) and 5′-CATGTGATTAAAACAAGTTTTCAAAGCCTTGAACTA-3′, and 5′-TGTATCACAATGTTAATGATATTGTTCCTGTCATG-3′ primers, respectively. The putative sites for miR-182 and -96 in FOXO1 were deleted from pFOXO1 using the primer 5′-AAATTTCATTACAATGAACTCACTACACCATATAAT-3′, and for miR-183 using primer 5′-CTGCTGTAGATAAGGACTTGGAAATTTCATTACAAT-3′.

The CYLD 3ʹ-UTR was amplified by PCR using the following primers: F 5′-GAGCTCGGCACCCATTGCCGGCA-3′ and R 5′-TCTAGAGGCAATCATTAGCTACA-3′. The purified PCR product was inserted into the pmirGLO dual luciferase plasmid to construct miRNA target expression vector (Promega, Madison, WI, USA). Moreover, 293T cells were planted into 96-well plates and transfected with the pmirGLO-CYLD 3′-UTR and either the miR-19a mimic or the control vector. After 48 h, the supernatants were collected, and the dual luciferase reporter kit (Promega, Madison, WI, USA) was used to detect the luciferase activity. Analysis of the firefly luciferase values was normalized to Renilla luciferase values to acquire the binding activity of microRNA.

The wild-type or mutant interacting sequences of miR-26a/b-5p in DLGAP1-AS1 sequence or in 3′-UTRs of IL-6, CDK8, and LRP6 were subcloned into the pmirGLO dual-luciferase plasmid (Promega, Madison, WI, USA). They were named as DLGAP1-AS1-WT/Mut, IL-6-3′-UTR-WT/Mut, CDK8-3′-UTR-WT/Mut, and LRP6-3′-UTR-WT/Mut. These vectors were co-transfected into HEK-293T cells with indicated transfection plasmids. After 48 h of co-transfection, relative luciferase activities were examined utilizing dual-luciferase reporter assay system (Promega).

LARP4 promoter was sub-cloned into pGL3 reporter vector (Invitrogen) to form pGL3-LARP4 promoter which was co-transfected into SKOV3 or A2780 cells with OE-circ_LARP4 or the empty vector. Circ_LARP4-WT/Mut or LARP4-WT/Mut was sub-cloned into pmirGLO dual-luciferase plasmid (Promega, Madison, WI, USA) to construct pmirGLO-circ_LARP4-WT/Mut or pmirGLO-LARP4-WT/Mut which was co-transfected with miR-513b-5p mimics or NC mimics into SKOV3 or A2780 cells. Luciferase activities were examined through Dual-Luciferase Reporter Assay System (Promega).

The 3’ UTR of PD1 was amplified from wild-type c57BL/6 lymphocyte cDNA using PCR with 10 μM PD1 3’ UTR forward and reverse primer:
Forward: 5’-ATATACTCGAGCCAGATTCTTCAGCCATTAGCATGCT
Reverse: 5’-GCGTGTCTAGATTTAAAGCTTTTGGTACCATTTAATTATAACGGGCT and Taq polymerase (Invitrogen). The amplified cDNA was separated on a 1.5% agarose gel and the QIAquick Gel Extraction Kit (Qiagen) was used to isolate the PD1 3’ UTR cDNA. The PD1 3’ UTR and pmirGLO Dual Luciferase Plasmid (Promega, USA) were cleaved with XhoI and XbaI restriction enzymes (New England Biolabs, Ipswich, MA) and allowed to ligate overnight at 16° C. The PD1 3’ UTR pmirGLO Dual Luciferase Plasmid was amplified in JM109 cells (Promega, Madison, WI) and extracted using the GeneJET Plasmid Miniprep Kit (Fermentas, Burlington, ON). The concentration of the plasmid was then measured using a NanoDrop ND-1000. Sequencing for confirmation of successfully ligation was done using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) in an Applied Biosystems 3730 DNA Analyzer (Applied Biosystems).