Pmirglo luciferase reporter plasmid

Fragments of the 3′UTR regions of putative miR-324 targets were amplified from cDNA, which was reverse transcribed from RNA extracted from C3H10T1/2 cells. PCR primers (Supplementary Table S4) were designed for In-Fusion HD cloning (Takara Bio Inc.) into the pmiRGLO luciferase reporter plasmid (Promega Corporation), which was digested with XhoI restriction endonuclease (New England Biolabs (UK) Ltd.). All predicted miR-324 binding sites were included in the amplified regions. Mutant Suox 3′UTR was synthesised as a gBlock (Integrated DNA Technologies), again designed for In-Fusion HD cloning. Mutant Cd300lf (at both predicted miR-324-5p binding sites) was produced by site-directed mutagenesis of the pmiRGLO-Cd300lf plasmid, using QuikChange Lightning (Agilent Technologies; mutagenesis sites shown in lower case; site 1 forward primer: 5′-AGGCAGGCTGCTTCAGGcAgGCTGTGTAAATCGTATC-3′; site 1 reverse primer: 5′-GATACGATTTACACAGCcTgCCTGAAGCAGCCTGCCT-3′; site 2 forward primer: 5′-AGCAGAAGGTGGAGGcAgGCAGAAGGAGTCAGG-3′; site 2 reverse primer: 5′-CCTGACTCCTTCTGCcTgCCTCCACCTTCTGCT-3′). Both mutant constructs contained mutations of 2 nucleotides within the predicted miR-324 binding sites. For all 3′UTR constructs except mutant Cd300lf, In-Fusion HD cloning was used, following the manufacturer’s protocol. All constructs underwent confirmation by Sanger sequencing (Source BioScience).

The mature sequences of miR-144-3p, located at chr17:28861533-28861618 (-), were obtained from miRBase². The putative target genes for miR-144-3p were predicted and overlapped by three algorithms: TargetScan³ (v7.0), miRanda⁴ (v3.3a), and RNAhybrid⁵ (v2.1.2). To generate the reporter vectors containing the potential binding sites of miR-144-3p, the DNA fragments of the 3′UTR of target gene PTEN (NCBI Accession Gene ID: 5728), were amplified from the extracted DNA of HCFs and then cloned into the pmirGLO luciferase reporter plasmid (Promega, United States). Two constructs of pmirGLO luciferase reporter plasmid were generated: MUT-PTEN (with mutation of part of miR-144-3p binding site sequence) and WT-PTEN (containing the wild-type miR-144-3p binding site sequence). 500 ng of the pmirGLO luciferase reporter plasmid and appropriate miRNA plasmid were co-transfected with lipofectamine 3000 (Invitrogen, United States) into HCFs. 24 h after transfection, the luciferase expression was determined using the Dual-Glo^TM Luciferase Reporter Assay Kit (Promega, United States) according to the manufacturer’s protocol. The pRL-TK vector (Promega, United States) containing Renilla luciferase was also co-transfected for normalization in all relevant experiments.

PRDX1 sequences containing wild-type (WT-Type) or mutated (MUT-Type) miR-431-5p binding sites were synthesized and inserted into pmiR-GLO luciferase reporter plasmid (Promega, WI, USA), respectively. LoVo cells were transfected using Lipofectamine 2000 (Invitrogen). PRDX1-WT or PRDX1-MUT were co-transfected with miR-431-5p-mimic or NC-mimic, respectively. After 24-h transfection, the luciferase activity was detected by the Glomax20/20 luminometer (Promega).

The wild type and mutant sequence of 3′ UTR of FOXO1 were cloned into the pmirGLO luciferase reporter plasmid (Promega) and sequenced to confirm the resulting plasmid by GenScript Biotech. hESCs were cultured in 24-well plates and transfected with the indicated plasmids. Cells were harvested, and the luciferase activities were analyzed after 48 h using a Dual-Luciferase Assay System (Promega) with a luminescence counter (Berthold Technologies) according to the manufacturer’s instructions. For normalization according to the transfection efficiency, firefly luciferase activity was normalized to the corresponding Renilla luciferase activity.

Mouse IL-10 3’-untranslated region (3’UTR) (+1 to +702) containing the miR-21-5p binding site was inserted into the NheI/XbaI site downstream of the luc2 gene of pmirGLO luciferase reporter plasmid (Promega, Madison, USA). Control and miR-21-5p agomir were purchased from Ruibo (Guangzhou, China). For dual-luciferase assay, CD4⁺CD8^-YFP⁺ Treg cells from the spleen of WT and Foxp3^Cre-YFPmiR-21^f/f mouse were flow sorted and transfected with mouse IL-10 3’UTR reporter plasmid alone or in combination with miR-21-5p agomir using Nucleofector Kit for mouse T cells (Lonza, Switzerland) per manufacturer’s instructions. Transfected cells were then cultured in the presence of 2 μg/ml anti-CD3, 2 μg/ml anti-CD28, 500 IU/ml IL-2, and 10 ng/mL TGF-β. After 48 h, the luciferase activities of the whole-cell lysate were analyzed using a dual-luciferase reporter assay system (Promega, USA). Data were normalized for transfection efficiency by dividing firefly luciferase activity by that of the Renilla luciferase.

The wild-type or mutated 3′-untranslated region (UTR) of CACNA1H was cloned into the pmirGLO luciferase reporter plasmid (Promega, Madison, WI, USA) and named wt-CACNA1H-3′UTR or mut-CACNA1H-3′UTR. The mutated sites are shown in Figure 1(a). The wt-CACNA1H-3′UTR or mut-CACNA1H-3′UTR was cotransfected into HEK293T cells with negative control (NC) mimic or miR-25-3p mimic. After 24 h, the luciferase activity was detected by using the dual-luciferase detection kit according to the manufacturer's instruction (KeyGEN, Nanjing, China).