Fig. 3.

Identification of functional microRNA (miRNA, miR) target sites and polymorphisms in a microRNA target site (PolymiRTSs) in HEK293 and HeLa cells. a Assessment of the relative effect of miRNAs on their predicted targets using luciferase reporter assays in HEK293 and HeLa cells. Luciferase constructs bearing the 3′ untranslated region (3′-UTR) of FERMT2, MS4A2, MS4A6A, and NUP160 were cotransfected with miR-4504, miR-585-3p, miR-3945, miR-626, miR-6867-3p, miR-6888-3p, miR-3976, miR-1185-1-3p, or scrambled (SCR) control miRNA. Each miRNA was cotransfected with the best predicted target allele, as indicated. Changes in lysate luciferase activity of the miRNA-transfected cells (relative to SCR control miRNA transfected cells) are shown. Negative and positive values indicate decreased and increased expression, respectively, compared with an SCR control miRNA. b Alignment between miR-4504, miR-3945, and miR1185-1-3p and 3′-UTRs of FERMT2, MS4A2, and NUP160. The physical consequences (creation and/or disruption of perfect seed matches) of minor allele PolymiRTS are indicated. (c and d) Luciferase assays showing the effect of rs7143400-G/T on the repressor activity of miR-4504 with regard to the 3′-UTR of FERMT2 in (c) HEK293 cells and (d) HeLa cells. (e and f) Luciferase assays showing the effect of rs2847655-T/C on the repressor activity of miR-3945 with regard to MS4A2 in (e) HEK293 cells and (f) HeLa cells. (g) and (h) Luciferase assays showing the effect of rs9909G/C on the repressor activity of miR1185-1-3p with regard to NUP160 in (g) HEK293 cells and (h) HeLa cells. *p < 0.05 by Mann-Whitney U test; ***p < 0.001 by Mann-Whitney U test; ns not significant by Mann-Whitney U test. The quoted data correspond to the average of the mean of at least three independent experiments performed in triplicate. The standard error of the mean is indicated on the graphs