ZEB1 and ZEB2 expression in UUO model mice.

<p><b>a</b>,<b>b</b>, Real time PCR analysis of ZEB1 or ZEB2 mRNA in kidneys <b>obstructed for 2,4,6 or 8</b> days. The data are normalized to beta-actin mRNA and shown as mean±S.E.(n = 8). <i>P</i><0.05: kidneys obstructed for 2,4,6 and 8 compared with 0 day <b>c</b>, Real time PCR analysis of ZEB1 or ZEB2 mRNA in <b>obstructed for 6 days</b> after i.v. administration of miR-200b precursor. The data are normalized to beta-actin mRNA and shown as mean±S.E.(n = 8). <i>P</i><0.05: <b>obstructed for 6 days</b> after i.v. administration of miR-200b precursor compared with negative control pre-miR.</p

miR-200 family members can ameliorate EMT and tubulointerstitial fibrosis in UUO model mouse kidneys.

<p><b>a</b>, Western blotting analysis confirms down-regulation of E-cadherin and up-regulation of N-cadherin during EMT. <b>b</b>,<b>c</b>, Western blotting of E-cadherin and N-cadherin in HK-2 cells treated with TGF-beta and transfected with control miR or miR-200 members precursors individually. In western blotting, each band has been quantitated and subjected to densitometry to determine if statistically significant difference exists between groups <b>d</b>, Changes in miR-200 family levels in UUO model mice kidneys, as measured by <i>TaqMan</i> qRT-PCR and normalized to U6 expression. The data are means from a representative time course experiment measured in triplicate and are presented as mean±S.E. <b>e</b>, Azan staining of intact Balbc mouse kidney, UUO day-6 kidneys after transfection of negative control pre-miR or miR-200b precursor. Fibrosis score; The tubulointerstitial fibrosis score in kidneys of miR-200b precursor injected group was significantly lower than of control group: <i>P</i><0.05, The data are means from experiment measured in triplicate and are presented as mean±S.E (n = 8) <b>f</b>, Immunofluorescence studies of vimentin and cytokeratin 18 in the kidneys of UUO day6 mice and UUO day6 mice injected by miR-200b precursor. miR-200b ameliorates up-regulation of vimentin and down-regulation of cytokeratin 18. <b>g</b>, real-time PCR confirms that miR-200b precursor reverses up-regulation of type I,III collagen and fibronectin of UUO day-6 kidneys. The data are normalized to beta-actin mRNA and shown as mean±S.E.(n = 8).</p

miR-200 family members target the transcriptional repressors ZEB1 and ZEB2.

<p>Normalized activity of luciferase reporter with the ZEB1 or ZEB2 3′UTR in HK-2 cells in the presence of co-transfected negative control pre-miR or miR-200 family individually. Luciferase activity was measured after 24 hours. The data are mean±S.E. of triplicates and are shown as the ratio of firefly to Renilla luciferase activity. <i>P</i><0.05: HK-2 cells in the presence of co-transfected miR-200 family compared with negative control pre-miR</p

Injected miR-200b precursor can be converted to mature miR-200b in kidneys.

<p><b>a</b>. Laser microscopy images of kidney, frozen sections at 24 hr after intravenously administration of Cy3 dye-labeled Pre-miR control. <b>b</b>. Changes in miR-200 family levels in Balbc mice kidneys 12, 24, 48 hours after intravenously administration of miR-200b precursor, as measured by <i>TaqMan</i> qRT-PCR and normalized to U6 expression. The data are means from experiment measured in triplicate and are presented as mean±S.E. <i>P</i><0.05: kidneys 12, 24 hours after intravenously administration of miR-200b precursor compared with 0 hour.</p