Rat bladder outlet obstruction leads to SMAD and extracellular-signal-regulated kinase (ERK) phosphorylation and to increased transcript levels of established transforming growth factor-β (TGF-β) targets.

<p>Phosphorylation of SMAD proteins (A) SMAD2/3 and (B) SMAD1/5/8 was measured by western blotting using phosphorylation-sensitive antibodies at various times following outlet obstruction. Gapdh was used as loading and normalization control. Protein expression was normalized first to Gapdh and then to the mean for the sham-operated controls. (C) Transcript levels for three established TGF-β targets are shown at 6 weeks of obstruction. The fold changes were large so the log₂ expression is shown on the y-axis. Data is from the mRNA microarray described in further detail in the text. (D) Time-course of ERK1/2 activation. All samples: n=6-8. * <i>p</i> < 0.05 versus sham. *** <i>p</i> < 0.001 versus sham.</p

MiR-29 depletion by genetic deletion of Dicer in smooth muscle increases detrusor stiffness in Ca²⁺-free conditions.

<p>Real-time quantitative polymerase chain reaction (<i>n</i>=5-7) for miR-29b (A), miR-29c (B) and elastin (Eln, C) in control (Ctrl) and Dicer knockout (KO) mouse bladders. Electron micrographs from control (D) and Dicer KO (E) detrusor. SMC: smooth muscle cell; N: neural varicosity. White arrowheads point to elastin fibrils. Scale bars represent 1 µm. Quantitative morphometry of collagen fibril diameters (F) and basal membrane thickness (G). (H) Western blots for Eln, Gapdh and Cav1 in detrusors from control and smooth muscle-specific Dicer knockout (KO) mice. Summarized data on Eln expression in control and Dicer KO bladders (I, <i>n</i>=12). (J) Passive circumference-stress curves in Dicer KO and control bladder strips (<i>n</i>=12). (K) Passive circumference-stress relations in control and elastase-treated rat detrusor strips (n=4).</p

Repression of miR-29 after outlet obstruction is associated with increased levels of miR-29 target proteins.

<p>(A) Western blots for eight miR-29 targets in sham-operated control bladders and at 6 weeks of obstruction. Gapdh and Cav1 were used as loading controls (<i>n</i>=6 throughout). (B) Relative fold change at 6 weeks of obstruction (vs. sham) of miR-29 target messenger RNAs (mRNA; black circles) and proteins (white circles). The fold change of mRNA compared to the fold change of the protein was significantly different throughout, except for Eln and Mybl2. MRNA expression is from the microarrays (<i>n</i>=6−8), and protein expression is from the western blots in A. Protein expression was normalized first to Gapdh and then to the mean for the sham-operated controls. (C) Immunofluorescence staining for collagen IV (col4a1, green) in sham-operated control and 6-wk obstructed detrusor. Scale bars represent 100 µm. The fluorescent pear-shaped profile in the center of the 6 weeks specimen represents the outline of a muscle bundle. Individual cell profiles are visible within the bundles. Nuclei are stained blue and the outer bladder surface is facing upward in both micrographs. </p

c-Myc accumulation in outlet obstruction.

<p>(A) Fold change (vs. sham) of c-Myc (Myc), Hdac3 and Ezh2 mRNAs in outlet obstruction and de-obstruction (n=6-8). (B) Inverse correlation between miR-29b and Myc mRNA level (both expressed relative to sham) in outlet obstruction and its reversal (n=6-8). The leftmost symbol represents 10 d obstruction, the rightmost symbol represents de-obstruction, and the symbols close to x=1 represent sham and 6 weeks of obstruction, respectively. Western blots and bar graph summaries showing increased c-Myc (C) and Ezh2 (D) levels in outlet obstruction (n=6 for both). (E) Western blots and bar graph summary showing increased phospho-NF-κB p105 in outlet obstruction (n=6). Blots for phospho-NF-κB p65 and IκB-α are shown without bar graph summaries as no significant differences were seen. </p

Reduced levels of the miR-132/212 targets Ache, MeCp2 and Pnkd in the bladder following outlet obstruction.

<p>Validated targets of miR-132/212 were examined at the protein level using western blotting at various times after surgical obstruction of the urethra (<b>A</b>). Summarized data (n = 6) for MeCP2, Ache and Pten is shown in panels <b>B</b> through <b>D</b>. Panel <b>E</b> shows double staining for MeCP2 (green) and DNA (blue) in control and obstructed (10 days) bladders. Panel <b>F</b> shows the percentage of MeCP2 positive nuclei in bladders from sham-operated and obstructed (10 days) rats (n = 6 and 7, respectively).</p

Smooth muscle-specific deletion of Dicer does not increase neostigmine-induced contraction or expression of Ache in the bladder.

<p>Panels <b>A</b> and <b>B</b> show contraction induced by the addition of the acetylcholine-esterase (Ache) inhibitor neostigmine in control and in Dicer knockout (KO) bladder strips. Force is given in mN (<b>A</b>) and relative to depolarization-induced contraction (<b>B</b>), respectively. Panel <b>C</b> shows the potentiation by neostigmine of twitches in response to electrical field stimulation (ΔNeo. twitch) in control and Dicer KO detrusor strips. Panels <b>D</b> and <b>E</b> show the mRNA and protein levels of Ache in control and Dicer KO bladders. Panels <b>F</b> and <b>G</b> show representative electron micrographs from sham-operated and obstructed (6 weeks) bladders. Neural varicosities are highlighted in red. Scale bars represent 1 μm. Panel <b>H</b> shows quantitative evaluation of synapse density in control and obstructed bladders. N = 6.</p

Transfection of miR-132/212 mimics and inhibitors affect MeCP2 expression and cell number in vitro.

<p>An initial dosing experiment performed in duplicate using human detrusor smooth muscle cells from one individual shows a reciprocal effect of miR-132 mimic and inhibitor on MeCP2 expression (<b>A</b>). The effect of 100 nM miR-132 mimic was confirmed using cells from three individuals (<b>B</b>). Panel <b>C</b> shows effect of miR-212 mimic and inhibitor (100 nM of each) on cell viability using the MTT assay. Panel <b>D</b> shows the effect of miR-212 mimic and inhibitor on cell number and panel <b>E</b> shows phase contrast light microscopic images of cells transfected with negative control (NC), miR-212 mimic and miR-212 inhibitor. The scale bar in <b>E</b> indicates 20 μm and n = 5 and 8 in <b>C</b> and <b>D</b>, respectively.</p

MiR-132/212 induction in the bladder correlates inversely with previously validated miR-132/212 targets.

<p>The normalized sum of miR-132 and miR-212 in bladders from sham-operated, obstructed (10 days and 6 weeks) and de-obstructed rats was correlated with the mRNA levels for <i>Mecp2</i> (<b>A</b>), <i>Ep300</i> (<b>B</b>), <i>Pnkd</i> (<b>C</b>), <i>Jarid1a</i> (<b>D</b>), <i>Sh3bp5</i> (<b>E</b>), <i>Ripk2</i> (<b>F</b>), <i>Foxo3</i> (<b>G</b>), <i>Pten</i> (<b>H</b>) and <i>Rasa1</i> (<b>I</b>). Expression data were obtained from microarray experiments (GEO accession GSE47080). Each symbol represents one rat and the 10d obstructed rats are represented by the rightmost symbols. The <i>p</i>-values obtained for the individual correlations are given at the bottom left in each diagram.</p

Primary antibodies used in the study are listed in the order that they appear in the figures.

<p>Primary antibodies used in the study are listed in the order that they appear in the figures.</p

Partial infravesical outlet obstruction increases expression of miR-132/212 in the urinary bladder.

<p>Rats were sham-operated (sham) or subjected to obstruction for 10 days (10d obs) and 6 weeks (6w obs). One group of rats were obstructed for 6 weeks and then re-operated to remove the obstruction. The obstruction-induced hypertrophy was then allowed to regress for another 10 days (de-obs). Bladders were harvested, RNA was isolated and microarrays were run (GEO accession GSE47080). Results in panels <b>A</b> and <b>B</b> are from these microarrays. Panels <b>C</b> and <b>D</b> show confirmation using qRT-PCR at 10 days of obstruction versus sham. *, **, and *** denote p<0.05, p<0.01 and p<0.001 in this and the following figures. N = 6–8 throughout.</p