Figure 3. Repression of miR-29 during outlet obstruction is associated with increased levels of miR-29 target messenger RNAs (mRNAs).

Gene expression was assessed (n=6−8) at 10 days and at 6 weeks following outlet obstruction using microarrays and compared with sham-operated controls and with rats where the obstruction had been removed for 10 days. (A) Relative increase (left vs. sham) and decrease (right vs. 6 weeks) of the top 50 mRNA targets of miR-29 when miR-29 was repressed at 10 days and when miR-29 recovered after de-obstruction (c.f. Figure 2A and B). The statistical comparison was made versus a theoretical value of 1 (no change) using Student’s t test. (B) Time courses of mRNA expression for six confirmed miR-29c targets (official symbols are given in the legend). Fold changes are versus sham. (C) Correlation between miR-29c and the mean fold change of the target mRNAs depicted in Figure 3B. (D) Western blots for miR-29 targets in human urinary bladder smooth muscle cells transfected with negative control and miR-29c-inhibitor (left row). Gapdh and caveolin-1 were used as loading controls. Cells were used in passages 3−5 and harvested 96 h after transfection. Targets shown were significantly changed (one-tailed Student’s t test) in independent replicates (n=3−5) using cells from one individual. Right row in D shows effect of miR-29c mimic. (E) Inverse correlation between fold change of miR-29c (vs. sham) and fold-change of Eln (vs. sham) in outlet obstruction/de-obstruction. (F) Inverse correlation between the mean fold change of miR-29b and miR-29c (both vs. sham) and fold-change of Sparc (vs. sham). In panels C, E and F the sham-operated controls are represented by the symbols at x=1, y=1 whereas 10 d obstructed bladders are represented by the leftmost symbols. The remaining two symbols represent 6 weeks obstructed and de-obstructed bladders, respectively.