Pmir glo luciferase reporter vector

The 3′-UTR fragment of CyclinG1 containing the miR-488-3p binding site was amplified by PCR. The fragment was inserted into the pmir-GLO luciferase reporter vector (Promega, USA) as described previously [17 (link)]. The PCR product fragments and pmirGLO plasmids were digested with SacI and XhoI. The digested fragments and vectors were ligated to construct recombinant plasmids. Then, recombinant plasmids were transformed into E. coli DH5α competent cells, coated on a LB plate containing 1% Ampicillin, and cultured overnight at 37°C. The recombinant monoclonal colonies were selected and amplified in LB liquid medium with 1% Ampicillin. The positive cloned plasmids were selected and sent to Sangon Biotech (Shanghai, China) for further sequencing verification. For the luciferase assay, miR-488-3p mimics were cotransfected with the luciferase reporters containing CyclinG1 3′-UTR or pmir-GLO luciferase reporter vector into HEK-293A cells using a transfection reagent (Vigofect, Vigorous Biotechnology, China). Forty-eight hours after transfection, luciferase activity was detected using the Dual-Luciferase Reporter Assay System (Promega).

The 3′-untranslated region (3′-UTR) of target genes and their mutant variant were synthesized and digested with SacI and XhoI to generate reporter vectors containing miRNA-binding sites (Shengong Co., China). The fragment was inserted into the pmir-GLO luciferase reporter vector (Promega, USA) using previously described methods [21 (link)]. For the luciferase assay, miR-154-5p mimics were cotransfected with the luciferase reporters containing Arsb-3′-UTR, mutant-Arsb-3′UTR, or pmir-GLO luciferase reporter vector into HEK-293A cells using a transfection reagent (Vigofect, Vigorous Biotechnology, China). Forty-eight hours after transfection, luciferase activity was detected using the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer's protocol.