Login to Access Video or Poster Abstract: MP82-19
Sources of Funding: SNSF Grant 320020_156161/1_x000D_ Velux Foundation Grant 895

Introduction

Bladder outlet obstruction (BOO) induces organ remodeling accompanied by changes in bladder function leading to lower urinary tract dysfunction (LUTD). MicroRNAs (miRNAs) may cause molecular changes in the bladder wall. Previously, using next generation sequencing (NGS) of mRNAs and miRNAs in human patients&[prime] biopsies we identified TNF-α as the top upstream regulator of signaling, potentially contributing to organ remodeling. Here we validated the NGS and pathway analysis in cell-based models using bladder smooth muscle (SM) and urothelial (UE) cells exposed to TNF-α.

Methods

TNF-α-responsive genes were selected based on LUTD patients&[prime] NGS data and in silico analysis. SMC and UE cells were treated with 10 ng/ml TNF-α 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, and results validated by qRT-PCR. Cell proliferation assay was performed to evaluate the proliferative effects of TNF-α.

Results

Primary SM and UE cells express TNF-α receptors TNFR1 and TNFR2 and respond to TNF-α treatment. NF_KB2, RelB and TNFAIP3 showed a progressive time- and concentration-dependent up-regulation, and responses were stronger in SM cells compared to UE. TNF-α treatment increased cell proliferation. MiRNA expression profiling identified 17 miRNAs altered in both SMC and UE cells. MiRNAs miR-146a-5p, -21-5p, -1260a, -183-5p, -22-3p, -199a-3p, -199b-3p were similarly regulated in patients and cell-based models. MiR-26b was significantly induced in UE and SMC, but down-regulated in BOO. There was a cell-type dependent difference in miRNA profiles, with SMC-specific miRNAs gown-regulated after TNF-α treatment, in accordance with the down-regulation of SM markers and loss of contractility in human patients. Transcriptome analysis of TNF-treated cells was carried out and expression levels of predicted targets of disease-relevant miRNAs identified.

Conclusions

Our results confirm an important role of TNF-α in the regulation of BOO-specific miRNAs, and identify miRNAs linking TNF-α signaling and fibrosis. Modulation of expression levels of TNF-α-regulated miRNAs in cell-based models of human bladder using miRNA-overexpression and inhibition will elucidate their role in organ remodeling and lead to novel therapeutic approaches for BOO-induced LUTD.

Funding

SNSF Grant 320020_156161/1_x000D_ Velux Foundation Grant 895