Pmirglo

To detect the bind ability between PCGEM1 (SOX11) and miR-590-3p, wide type full-length sequences of PCGEM1 (SOX11) and the mutant-type PCGEM1 (SOX11) were cloned into the firefly luciferase gene reporter vector, pmirGLO (Promega, Madison, WI, USA). The plasmid was synthesized by Invitrogen. The pmirGLO-PCGEM1(SOX11)-Wt or pmirGLO-PCGEM1 (SOX11)-Mut was co-transfected with miR-590-3p mimics (inhibitor) or NC mimics (inhibitor) (RiboBio, Guangzhou, China) into HEK293T cells. After 48-hour of transfection, the luciferase assay was performed with the dual-luciferase reporter assay system kit (Promega) according to the manufacturer’s guidance.

To investigate the interaction between SERPINC1 and miR-200c-3p, wide type or mutant 3′-UTR of SERPINC1 was cloned into the firefly luciferase gene reporter vector pmiRGLO (Promega, Madison, WI, USA). The plasmid was synthesized by Invitrogen. The pmiRGLO-SERPINC1-Wt or pmiRGLO-SERPINC1-Mut was co-transfected with miR-NC or miR-200c-3p inhibitor (RiboBio, Guangzhou, China) into HEK293 cells using Lipofectamine 3000 (Invitrogen). The blank cells were regarded as control cells. Forty-eight h post transfection, cells were harvested. Luciferase assay was performed using the Dual Luciferase Reporter Assay System (Promega) under the protocol of manufacturer. The relative luciferase expression equaled the expression of the Renilla luciferase divided by the expression of the firefly luciferase [37 (link)]. Transfection was repeated for at least 3 times.

The 293 T cell line was used in this study. Cells were maintained in Dulbecco's modified Eagle’s medium (DMEM) (Gibco, Waltham, USA) supplemented with 10% fetal bovine serum (Gibco), penicillin and streptomycin and maintained in an incubator with 5% CO₂ at 37 °C. The predicted binding sites were cloned and then inserted into the pmirGLO (Promega, Madison, USA) reporter vector. For reporter assays, 150 ng of pmirGLO reporter vector and 50 nM miR-275 mimic (RiboBio, GuangZhou, China) were co-transfected into 293 T cells using Lipofectamine 2000. No-mimic treatment cells were used as a blank control, and pmirGLO-Vg vector only cells were used as the negative control. Firefly and Renilla luciferase activities were measured 48 h post-transfection by the Dual-Luciferase Reporter Assay System (Promega). First, 100 μl of luciferase assay reagent II was added to each well, Firefly luciferase activities was measured, and 100 μl Stop&Glo reagent was loaded into each well. Then, Renilla luciferase activities were measured. Firefly luciferase in the pmirGLO vector was used for normalization of Renilla luciferase expression. Treatments were assessed in triplicate, and transfections were repeated three times. Firefly luciferase activities were divided by Renilla luciferase activities. Finally, the ratio between Renilla and Firefly luciferase activities was calculated for each experiment.

Human FRMD6 3'UTR region and its sequence with a mutation of the miR-93-5p seed sequence were inserted into a firefly/Renilla luciferase reporter vector (pmirGLO) obtained from RiboBio. PC3 cells were transfected with pmirGLO-FRMD6-WT or the mutant construct, and miR-mimic-NC or miR-93-5p-mimic by using Lipofectamine 3000 (Invitrogen). 48 h after transfection, Dual Glo Luciferase Reporter Gene Assay Kit (Yeasen, China) was used to examine the luciferase activity.