Dual glo luciferase detection system

The predicetion of the binding sites of miR-499-5p and TGFβ-R1 was performed by TargetScan 7.2 (http://www.targetscan.org/). To verify if TGFβ-R1 is the target gene of miR-499-5p, the fragment of the 3′UTR region of wide-type (Wt) and mutant-type (Mut) TGFβ-R1 were PCR amplified, using the following primers (binding sites are underscored, restriction sites of XhoI and NotI are in italics), respectively: TGFβ-R1 Wt forward, 5′-CCGCTCGAGCTGAATTCTAAATCTACCTCAAGGATCTAAGGAGCGGCCGCTAAACTAT-3′, and reverse, 5′-TAAGAATGCGGCCGCTCCTTAGATCCTTGAGGTAGTTTAGAATTCAGCTCGAGCGG-3′. TGFβ-R1 Mut forward, 5′-CCGCTCGAGCTGAATTCTAAATCGACAGCTAGGATCTAAGGAGCGGCCGCTAAACTAT-3′, and reverse, 5′-ATAAGAATGCGGCCGCTCCTTAGATCCTAGCTGTCGTTTAGAATTCAGCTCGAGCGG-3′. Afterwards, the transcript was cloned into the psiCHECK2 vector (Promega). Subsequently, a total of 2 × 10⁵ 293T cells per well were inoculated and co-transfected with either miR-499-5p mimic or miR-499-5p NC using Lipofectamine 2000® (Invitrogen), according to the manufacturer’s guidelines. After transfection for 48 h, luciferase activity was assayed in triplicate according to the manufacturer’s instructions of a DUAL-GLO® Luciferase Detection System (Promega).

The wild-type (WT) EPHA2 promoter sequence containing binding sites with miR-451a was cloned into pmirGLO luciferase reporter vector (Promega), and mutant (MT) EPHA2 promoter sequence without binding sites was constructed into the same vector to generate report plasmids EPHA2-WT/EPHA2-MT. The 293 cells were co-transfected with 50 nM miR-451a mimic or NC mimic and 500 ng/mL reporter plasmids for 2 days. The luciferase intensity was assessed by a Dual-Glo® luciferase detection system (Promega).

To verify the targeting of miR-183-5p with CTSB, a CTSB 3′' untranslated region fragment with wild-type (WT) or mutant-type (MUT) miR-183-5p putative binding site was cloned via PCR. Insertion of the PCR products was into the pMir-Glo vector (synthesized via Shanghai GenePharma Co., Ltd.). Seeding of HEK293 cells was into 96-well plates at a density of 1 × 10⁵ cells/well. Then, cotransfection of miR-183-5p mimic, mimic NC, and luciferase reporter vector was into HEK293 cells, respectively, adopting Lipofectamine 2000. After transfection of 24 h, successive measurement of the luciferase activity of the firefly and the kidney was performed adopting the Dual Glo luciferase detection system (Promega).