Dysregulation of microRNAs (miRNAs) adds to molecular changes in the bladder wall during bladder outlet obstruction (BOO). However, the nature of miRNA activity remains elusive, complicating the selection of potential miRNA drivers of organ remodeling for therapeutic purposes. Previously, using next Generation sequencing (NGS) of mRNAs and miRNAs in human patients’ biopsies and cell based systems we identified and validated TNF-α as the top upstream regulator of signaling in obstructed bladders. In the patients’ biopsies, the up-regulated targets of the inhibited miRNAs contributed to the majority of activated signaling pathways. Here we sought to investigate the specific contribution of TNF-a-induced and -inhibited miRNAs to aberrant signaling in the smooth muscle cells (SMC), leading to cell proliferation and bladder fibrosis.
SMC cells were treated with TNF-a, and RNA isolated. Regulation of TNF-α-induced genes was studied by qRT-PCR and comprehensive transcriptome analysis performed by NGS. NanoString nCounter miRNA assays were used to Profile miRNAs. miRNAs and anti-miRs were expressed in SMCs using lentiviral vectors. NFkB-luciferase assays were performed to determine the effect of miRNAs on TNF-a signaling.
Compensatory overexpression of SM-specific, TNF-a-inhibited miR-199a-5p significantly reduced NFkB-driven luciferase gene expression via down-regulation of is direct targets IKKB, MAP3K5, NFKB1 and PIK3CD. siRNA-mediated knockdown of IKKB or MAP3K5 mimicked the effect of miR-199a-5p overexpression on TNF-a signaling. To investigate the role of other TNF-a-inhibited miRNAs on SM remodeling, transcriptome analysis of TNF-treated cells was carried out and expression levels of predicted targets of disease-relevant miRNAs examined. We show that 629 targets of 15 down-regulated miRs were up-regulated, whereas only 352 targets of 13 elevated miRs were decreased, indicating the higher functional impact of down-regulated miRNAs. Indeed, 269 elevated mRNA targets of down-regulated miRNAs are implicated in regulation of 80% of all TNF-induced pathways. Top pathway elements were targeted specifically by miR-199a-5p, miR-424-5p and miR-149-5p. All these miRNAs are also reduced in BOO patients’ biopsies, making them attractive candidates for therapeutic intervention.
Our results indicate that compensatory up-regulation of miR-199a-5p and other miRNAs, inhibited by TNF-a might have a beneficial effect reducing the proliferative and fibrotic changes in BOO bladders. The importance of the targets of down-regulated miRNAs for signaling might mean that the therapeutic efforts should concentrate on finding the pharmacological ways to enhance their expression to counter-balance fibrotic bladder remodeling.