EGLN2 as a novel downstream target of miR-205.

<p>(A) Quantitative real-time RT-PCR analysis revealed that the expression of EGLN2 was increased by miR-205 inhibition, but not significantly altered by overexpression of miR-205 compared to negative control transfected HK-2. No changes were seen in expression levels of EGLN1 (PHD2) or EGLN3 (PHD3). The data represent the means ± S.E. of triplicate analysis from three independent experiments. *P<0.05 by Dunnett’s multiple comparison test. (B) Western blot analysis showed that EGLN2 was also increased at protein level by miR-205 inhibition. The result of densitmetry represent the means ± S.E. of triplicate analysis from two independent experiments.**P<0.01 by standard <i>t</i>-test. (C) Quantitative real-time RT-PCR analysis revealed that the expression level of EGLN2 was increased in HK-2 exposed to hypoxia-reoxygenation or TUN, but these increases were supressed by miR-205 overexpression (▪) compared to negative control transfected HK-2 (□). *P<0.05 by Tukey’s multiple comparison test. (D) EGLN2 (transcript variant 1) contains the predicted binding site for miR-205 in its 3′-UTR. The predicted pairing of the mRNA target region (top) and miRNA (bottom) is as indicated, wherein solids line indicates hydrogen base pairing and the dotted line indicates Watson-Crick/wobble base pairing. The shaded sequence indicates the seed region of miR-205. (E) Mutant 3′-UTR sequence that abolished binding to miR-205. (F) HK-2 cells were transiently cotransfected with a luciferase reporters expressing EGLN2 3′-UTR or mutant EGLN2 3′-UTR miRNA target sequence and miR-205 mimic, miR-205 inhibitor, or negative control. At 24 hours posttransfection, firefly luciferase activities were measured and normalized with <i>Renilla</i> luciferase activities. Relative luciferase activity was increased by miR-205 inhibition when compared with control, while mutant EGLN2 3′-UTR had no effect on luciferase activity, indicating that miR-205 acts on the 3′-UTR of the EGLN2. Though transfection of HK-2 with EGLN2 3′-UTR vector along with miR-205 overexpression tended to decrease luciferase activity, this change was not sicnificant. The data represent the means ± S.E. of triplicate measurements from two independent experiments. *P<0.05 by Tukey’s multiple comparison test. (G) When transiently transfected HK-2 with EGLN2 3′-UTR vector were exposed to oxidative or ER stress, the relative luciferase activities were significantly increased, and these increases were canceled with mutant EGLN2 3′-UTR vectors. Furthermore, co-transfection of EGLN2 3′-UTR vector with miR-205 mimic partially restrained these stress-induced increases of luciferase activities. The data represent the means ± S.E. of triplicate measurements from two independent experiments. *P<0.05 by Tukey’s multiple comparison test.