Figure 7. miR-17∼92 deletion results in improved expression of mitochondrial and metabolism-related gene networks.

RNA-Seq analysis was performed to compare mRNA expression profiles. (a) Top differentially regulated pathways in 21-day-old Pkd1^F/RCDKO compared with Pkd1^F/RCSKO kidneys, and 21-day-old Pkd2-miR-17∼92KO compared with Pkd2-KO kidneys are shown. (b) Ingenuity pathway analysis software was used to identify the upstream regulators (URs) that may be responsible for the gene expression changes observed after miR-17∼92 deletion in ADPKD models. Top 10 (based on z scores) differentially expressed gene networks and their associated URs in Pkd1^F/RCDKO compared with Pkd1^F/RCSKO kidneys are shown. These networks were also differentially regulated in Pkd2-miR-17∼92KO compared with Pkd2-KO kidneys. Positive z scores (shades of orange) indicate activation whereas negative z scores (shades of blue) indicate inhibition of the gene networks. The P values (shades of purple) represent the level of statistical confidence for the prediction that the differentially expressed gene network is indeed regulated by the indicated UR. Large interconnected gene networks controlled by URs PPARα, PPARg and PPARGC1a were predicted to be activated after miR-17∼92 deletion in both ADPKD models. (c) RNA-Seq data were intersected with high-probability miR-17∼92 targets predicted by TargetScan. This analysis identified Pparα and 24 other common putative miR-17∼92 targets in the context of ADPKD. Expression of these genes was decreased (shades of green) in single knockout (SKO) compared with their respective control (Ctl) kidneys. In contrast, the expression of these genes was increased (shades of red) in double knockout (DKO) compared with their respective SKO kidneys. SKO indicates either Pkd1^F/RCSKO or Pkd2-KO, whereas DKO indicates either Pkd1^F/RCDKO or Pkd2-miR-17∼92KO kidneys. The circles indicate predicted binding sites for the various miRNA families derived from the miR-17∼92 cluster. (d) Q-PCR analysis showing Pparα expression in the indicated mouse models or cell lines. (e) Western blot showing increased PPARα expression in Pkd1^F/RCDKO compared with Pkd1^F/RCSKO kidneys. (f) PPARα antibody staining revealed that PPARα expression was increased in cyst epithelia of Pkd1^F/RCDKO mice compared with Pkd1^F/RCSKO mice. PPARα expression was decreased in mIMCD3 cells treated with miR-17 mimic compared with scramble mimic. Error bars indicate s.e.m. *indicates P<0.05, ns indicates P>0.05. One-way ANOVA, Tukey's multiple comparisons test, Student's unpaired t-test. Scale bars, 50 μm (f, top panel) and 20 μm (f, bottom panel).