Pmirtarget

The IGF1R 3′ UTR clone in pMirTarget was obtained from Origene. The IGF1R 3′ UTR reporter was constructed by amplifying the endogenous IGF1R 3′ UTR from the Origene. XhoI and ApaI sites were added to the 5′ and 3′ ends of the fragment during the preceding PCR reaction and cloned into the XhoI and ApaI site on the Rr-luc-6XCXCR4 Renilla luciferase vector (Addgene). To make the IGF1R 3′UTR mutant construct, site-directed mutagenesis was used to delete 99-105, 2619-2626, and 6661-6667 regions, corresponding to the predicted let-7b binding sites. A firefly luciferase vector was used as transfection and normalization control in all luciferase assays. Constructs were sequence verified before being used in experiments.

MiRNA target binding was validated by Dual Luciferase Assay (Promega, Madison, WI, USA). DNA vectors containing clones of target 3′UTRs (pMiRTarget; Origene, Rockville, MD, USA) were co-transfected with miRNA or control mimics into LX-2 cells following manufacturer’s recommendations. Luciferase activities were measured 48 h after transfection with a bioluminescence plate reader.

The PPP1R1C 3′ UTR clone in pMirTarget was obtained from Origene. The PPP1R1C 3′ UTR reporter was constructed by amplifying the endogenous PPP1R1C 3′ UTR from the Origene. XhoI and ApaI sites were added to the 5′ and 3′ ends of the fragment during the preceding PCR reaction and cloned into the XhoI and ApaI site on the Rr-luc-6XCXCR4 Renilla luciferase vector (Addgene). To make the PPP1R1C 3′UTR mutant construct, site-directed mutagenesis was used to delete 2165-2172 region, corresponding to the hsa-miR-182 binding site. A firefly luciferase vector was used as transfection and normalization control in all luciferase assays. The PPP1R1C plasmid encoding the coding sequence was obtained from BioClone Inc. (USA). Constructs were sequence verified before being used in experiments.

Cells were seeded into 96-well plates and co-transfected using TransIT-X2 (Mirus, Madison, WI, USA) with microRNA mimic (Thermo Scientific) or Anti-miR (Thermo Scientific) along with 50 ng of pMirTarget (Origene, Rockville, MD, USA) housing the 3′UTR sequence of TET1 that contained either a wild-type or mutant version of the miR-29b binding site. Luciferase and RFP expression were measured using the Steady Glo Luciferase kit (Promega, Madison, WI, USA) and a Tecan microplate reader.

miRNA activity was quantified by co-transfecting 293T cells (ThermoFisher, Leicestershire, UK) with Lipofectamine 2000 (ThermoFisher, Leicestershire, UK), a human GAB1 3′-UTR reporter plasmid (containing 3770 bp immediately downstream of the end of the GAB1 ORF cloned into pMirTarget, Origene), a control Renilla luciferase plasmid (Promega) and pre-miR miRNA mimics or pre-miR control 1 (ThermoFisher, Leicestershire, UK). Luciferase activity was quantified at 24 h using the dual-glo luciferase assay system (Promega, Southampton, UK), normalised using Renilla luciferase values from the same well and normalised values for control transfected cells (no pre-miR) were set to 1.0. Cell line identity was routinely confirmed using short tandem repeat analysis (Powerplex 16 System, Promega, Southampton, UK) and absence of mycoplasma was confirmed using the Mycoplasma PCR detection kit (Applied Biological Materials, Richmond, Canada).

pMirTarget, pMirTarget-wt-PPARγ-3′-UTR, and pMirTarget-wt-ZO-1-3′-UTR were obtained from OriGene Technologies (USA). By using Lipofectamine™ 3000 (Invitrogen, USA), BEAS-2B cells were transfected with the miR-27a mimic and miR-23a mimic, as well as Cy3-dye-labeled negative pre-miR control (Thermo Fisher Scientific, USA), and reporter vectors. A Dual-Luciferase Assay kit (Promega, USA) was used to detect luciferase activity according to the manufacturer’s instructions.

293T cells were co-transfected in triplicates in 96 well plates, 24 h post plating, with 50 ng of each luciferase reporter clones and 20 nM of either siNT1 or siGGGGGC using Lipofectamine 2000 (Thermofisher). The control plasmid, pMirTarget (#PS100062); PES1 human clone (#SC205900, NM_014303); POLR2K human clone (#SC208896; NM_005034); POLR2E human 3UTR clone (#CW306737, NM_00131623); EIF5A human 3UTR clone (#SC208525, NM_001143760); Mutant 3′ UTR clones for PES1 (#CW306514), POLR2K (#CW306515), POLR2E (#CW306738), and EIF5A (#CW306739) were all purchased from Origene. Mutant plasmids were generated by synthesizing 3′ UTR sequences in which the three GCCCCC seed matches in the 3′ UTR of PES1, one in 3′ UTR of POLR2K, three in POLR2E 3′ UTR and 5 seed matches in EIF5A 3′ UTR was each changed to TGCAAA, and this altered sequence was each inserted into the pMirTarget construct by Origene. After 48 h, transfection efficiency was determined by measuring Red Fluorescence Protein (RFP) expression of the luciferase reporter plasmid followed by measurement of luciferase activity after lysing cells with Bright-Glo Luciferase Assay System (Promega #E2610), both using Biotek Cytation 5 plate reader. Data are shown as relative luciferase activity normalized to RFP expression.