In vitro target assay of microRNAs on the SERPINB3 transcript.

[A] Diagram of miR-101, miR-1668 and miR-1681 binding sites in SERPINB3 3′-UTR. [B] Expression vector maps for eGFP with SERPINB3 3′-UTR and mutated SERPINB3 3′-UTR and Ds-Red with each miRNA. The wild-type (WT) and mutants of 3′-UTR of the SERPINB3 transcript were subcloned between the eGFP gene and the polyA tail to generate the fusion construct of the GFP transcript following the miRNA target 3′-UTR (pcDNA-eGFP-3′UTR) (top and middle panel) and the miRNA expression vector was designed to co-express DsRed and each miRNA (pcDNA-DsRed-miRNA) (bottom panel). [C] After co-transfection of pcDNA-eGFP-3′UTR for the SERPINB3 transcript and pcDNA-DsRed-miRNA for the miR-101, miR-1668 and miR-1681, the fluorescence signals of GFP and DsRed were detected using fluorescent microscopy. [D] The rate of inhibition of eGFP expression from miRNA modulation was calculated by fluorescence-activated cell sorting (FACS). Solid bars represent WT of SERPINB3 3′-UTR and empty bars show the mutant of SERPINB3 3′-UTR for each miRNA. Error bars indicate the standard error of triplicate analyses. The asterisks denote statistically significant differences between WT vs. mutants (***P<0.001).