Μεσαίες πόλεις : προοπτικές ανάπτυξης - διαφανείς τάσεις στο ελληνικό αστικό σύστημα

<p>HR3∶ 16 hours hypoxia followed by 3 hours reoxygenation.</p

miRNA expression profiles under ER stress.

<p>(A) Volcano plots showing the global changes in miRNA expression by ER stress. Points with a fold-change more than 1.2 or less than 0.8 with statistical significance are shown in dark gray. (B) Real-time RT-qPCR analysis revealed equally increased expression of GRP78 in HK-2 exposed to TUN, or THG, indicating that ER stress induced by each chemical was equivalent. The data represent the means ± S.E. of triplicate analysis from two independent experiments. *P<0.05 by Tukey’s multiple comparison test.</p

Suppression of miR-205 increased oxidative stress.

<p>(A) Representative figure of the result of FACS. Formation of intracellular ROS was confirmed using CM-H2DCFDA, a dye that emits green fluorescence on reaction with ROS. miR-205-inhibited HK-2 cells were exposed to H₂O₂ in a short time as an oxidative stress inducer. The fluorescence value of miR-205-inhibited HK-2 was higher than the control both before (uncolored area) and after (gray area) induction by H₂O₂. ROS-positive expression boundaries are marked by horizontal lines (M1) on the histogram plots. (B) Changes in the proportion of ROS-positive cells by miR-205 inhibition before and after H₂O₂ treatment. Intracellular ROS was significantly increased in miR-205-inhibited HK-2 (▪) compared to negative control transfected HK-2 (□) even before the cells were exposed to H₂O₂. The data represent the means ± S.E. of duplicate analysis from three independent experiments. *P<0.05 by Tukey’s multiple comparison test. (C) Western blot analysis of CEL showed that the intensity of bands with a molecular weight corresponding to 32, 23, and 17 kDa (black arrows) were increased in miR-205-inhibited HK-2 compared with control inhibitor transfected HK-2, suggesting that intracellular ROS was increased by down-regulation of miR-205.</p

miRNA expression change in HK-2 exposed to 10 hrs reoxygenation after hypoxia.

<p>HR10∶ 16 hours hypoxia followed by 10 hours reoxygenation.</p

Expression changes of HIF and ATF4 modulated by miR-205.

<p>(A) Quantitative real-time RT-PCR analysis revealed that the expression level of both VEGF and GLUT1, the down stream targets of HIF, were decreased by miR-205 down-regulation, but not significantly changed by miR-205 up-regulation. The data represent the means ± S.E. of triplicate measurements from two independent analysis. **P<0.01 by Dunnett’s multiple comparison test. (B) HK-2 cells were transiently cotransfected with a HRE-luciferase reporter and miR-205 inhibitor or negative control. At 24 hours posttransfection, firefly luciferase activities were mesured and normalized with <i>Renilla</i> luciferase activities. Relative luciferase activity was significantly decreased by miR-205 inhibition, suggesting that HIF was indirectly regulated by miR-205. The data represent the means ± S.E. of triplicate measurements from two independent analysis. *P<0.05 by standard <i>t</i>-test. (C, D) Western blot analysis of HIF-1α (C) and HIF-2α (D) revealed that both HIF proteins were significantly decreased by down-regulation of miR-205. The results of densitometry represent the means ± S.E. of triplicate measurements from two independent analysis. **P<0.01 by standard <i>t</i>-test. (E) Western blot analysis of ATF4 revealed that down-regulation of miR-205 led to significant decrease in nuclear ATF4 accumulation, indicating that ATF4 was also indirectly regulated by miR-205. The results of densitometry represent the means ± S.E. of triplicate measurements from two independent analysis. **P<0.01 by standard <i>t</i>-test.</p

miRNA expression change in HK-2 exposed to ER stress.

<p>TUN: tunicamycin of 2 µg/ml for 24 hours.</p><p>THG: thapsigargin of 0.5 µg/ml for 24 hours.</p

miR-205 contributes to both signalings triggered by oxidative and ER stresses in renal tubular cells.

<p>When cells are exposed to oxidative stress or ER stress, maladaptive down-regulation of miR-205 and subsequent up-regulation of EGLN2 occurs. ATF4 and HIF, which are negatively regulated by EGLN2, are supposed to contribute to the downstream suppression of anti-oxidant enzymes.</p

Change in cell survival under oxidative and ER stresses in miR-205-inhibited HK-2.

<p>(A) Inhibition of miR-205 in HK-2. Real-time RT-qPCR analysis revealed that transfection of an LNA-miR-205 inhibitor into HK-2 for 24 hours significantly inhibited miR-205 expression. The data represent the means ± S.E. of triplicate analysis from three independent experiments. **P<0.01 versus control siRNA transfected HK-2 by standard <i>t</i>-test. (B) In miR-205-inhibited HK-2, the cell number was significantly fewer at 24 and 48 hours after transfection than that of with HK-2 transfected with negative control. In contrast, up-regulation of miR-205 had no effects on the cell number. The data represent the means ± S.E. of triplicate counts from three independent experiments. *P<0.05 versus negative control transfected HK-2 at each time point by standard <i>t</i>-test. (C) Representative pictures of HK-2, 24 hours after transfection with miR-205 inhibitor or control inhibitor. Although cell growth was repressed, cell morphology of miR-205-inhibited HK-2 was not different from control siRNA transfected HK-2. (D) LDH release assay showed that modulation of miR-205 did not affect cell death, suggesting that the decrease in cell number in miR-205-inhibited HK-2 was not due to cell injury, but rather to repression of cell growth. The data represent the means ± S.E. of triplicate analysis from three independent experiments. Statistical analysises were performed by Tukey’s multiple comparison test. (E,F) Trypan blue exclusion assay showed the cell viability changes in miR-205 modulated HK-2 (▪) exposed to hypoxia-reoxygenation (0.1% O₂ for 16 hours followed by 6 hours reoxygenation), H₂O₂ (1000 µM for 6 hours) or TUN (2 µg/ml for 24 hours) compared to HK-2 transfected with negative control (□). Down-regulation of miR-205 led to significant decrease in cell viability by each stimulation (E), wheares up-regulation of miR-205 vice versa (F). Cell viabilities were not changed without any stimulations. The data are the means ± S.E. of triplicate counts from three independent experiments. *P<0.05, **P<0.01 by standard <i>t</i>-test.</p

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.</p

miRNA expression change in HK-2 exposed to thapsigargin.

<p>THG: thapsigargin of 0.5 µg/ml for 24 hours.</p