Pmirglo dual luciferase reporter vector

The wild-type or mutant 3′ UTR of NFATC4 mRNA, containing the miR-133b and miR-532-3p seed sequence targeting sites, was biosynthesized (Genewiz, Suzhou, China) and modified to include Xho I and Sal I restriction enzyme sites. Subsequently, all the obtained sequences were treated with restriction enzymes, Xho I and Sal I, and ligated to the pmirGLO dual-luciferase reporter vector (Promega, Madison, AL, USA) using T4 DNA ligase (TaKaRa, Dalian, China). The plasmids were sequenced to verify the correct insertion (Genewiz, Suzhou, China). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used to co-transfect HEK293T cells with the wild-type or mutant 3′ UTR luciferase reporter plasmids, and the mimics NC, miR-133b mimic, and miR-532-3p mimic (RiboBio Co., Guangzhou, China). The cells were harvested 48 h after transfection, and the luciferase activities were measured using a Dual-Glo Luciferase Assay System (Promega; Madison, WI, USA). Firefly luciferase was used as the normalization control.

The wild-type (WT) or mutant-type (MUT) circGRAMD1B fragment and the 3′-untranslated region (UTR) of PTEN and p21 were subcloned downstream of the luciferase gene within the pmirGLO dual-luciferase reporter vector (Promega, Madison, WI, USA). HEK-293T cells grown in a 96-well plate were cotransfected with plasmids containing the 3′-UTR of the wild-type or mutant fragment from circGRAMD1B, PTEN, and p21 and its paired miR-130a-3p mimics using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 48 h of transfection, firefly and Renilla luciferase activities were measured by using a dual-luciferase reporter assay system. The ratios of luminescence from firefly to Renilla luciferase were calculated, and each assay was repeated in 3 independent experiments.

For E2F1 overexpression vector, the E2F1 coding sequences were amplified and inserted into the pcDNA-3.1 vector (Invitrogen) using the XhoI and EcoRI restriction sites. For pmirGLO dual-luciferase miRNA target reporter vector, the E2F1 3′ UTRs were amplified and inserted into the pmirGLO dual-luciferase reporter vector (Promega) using the DraI and SalI restriction sites. E2F1-3′ UTR mutant plasmid was generated by changing the binding site of miR-20a-5p and miR-20b-5p from CACTTT to AGAGCG. PCR amplification was performed to do the mutagenesis and DpnI digestion to remove the parental DNA. For miR-17~92 and miR-106a~363 promoter reporter plasmids, four fragments of the miR-17~92 and miR-106a~363 promoters were amplified using the primers listed in Supplementary File 2. Then the PCR products of Reporter-1 and Reporter-2 were digested with KpnI and SmaI, the PCR products of Reporter-3 was digested with SacI and SmaI, and the PCR products of Reporter-4 was digested with KpnI and XhoI. After digestion, the products were inserted into the pGL3-basic reporter vector (Promega) to create the reporters of pGL3-R1, pGL3-R2, pGL3-R3 and pGL3-R4.

HOTTIP or PGK1 3’UTR segment covering wild-type (Wt) and mutant-type (Mut) miR-30b-3p-binding site was inserted into pmirGLO dual-luciferase reporter vector (Promega, MI, USA) to obtain pmirGLO-HOTTIP-Wt/Mut and pmirGLO-PGK1-Wt/Mut. The WT (or Mut) pmirGLO luciferase reporter gene vector of HOTTIP (or PGK1 3’UTR) and miR-30b-3p mimic (or mimic NC) were co-transfected into cells with Lipofectamine 3000 (Invitrogen). The luciferase activity was analyzed using a dual-luciferase reporter gene detection system (Promega).