MiR-138 decreases S100A1 gene expression in Endothelial Cells.

<p>A) EA.hy926 ECs were co-transfected with the S100A1–3′UTR luciferase reporter gene and either a MiR-138 mimic or scrambled control mimic. Luciferase activity in cell lysates was measured 24 h later and is reported as relative light units (RLU) normalized to untreated cells. *, P<0.01 vs ctr. mimic. The experiment was done 3 times, each in triplicate. Expression of a luciferase reporter linked to the control 3′UTR was not changed by the mimic treatment (not shown). B) Primary HMVEC were transfected with the MiR-138 mimic for 1 or 3 days or control mimic for 3 days. Cell extracts were immunoblotted for S100A1 or β-actin (as loading control). Transfection with the control mimic did not change S100A1 levels compared to untransfected HMVECs (not shown). A representative immunoblot is shown. The experiment was performed 3 times. Expression of S100A1 was normalized to β-actin. *, P<0.05 vs ctr. Mimic. C) EA.hy926 ECs were transfected with either the wild-type (WT) S100A1–3′UTR luciferase reporter gene or a S100A1–3′UTR with deletion of the 22 nucleotide putative MiR-138 target site (ΔMiR138). 24 h later cells were subjected to chemical hypoxia (250 µmol/L CoCl₂). Luciferase activity in cell lysates was reported as relative light units (RLU) normalized to WT untreated cells. *, P<0.01 vs untreated. The experiment was done 3 times, each in triplicate.