MicroRNAs (miRNAs) are small non-coding regulatory RNAs that are altered in patients with lower urinary tract dysfunctions (LUTD). miRNAs regulate gene expression and may cause molecular changes in the bladder wall during bladder outlet obstruction (BOO). Previously, using next-generation sequencing (NGS) of messenger RNAs and miRNAs in human patients’ biopsies, we identified tumour necrosis factor alpha (TNF-α) as the top upstream regulator of signalling, potentially contributing to organ remodelling. Here we validated the NGS and pathway analysis in cell-based models using bladder smooth muscle (SM) cells and urothelial (UE) cells exposed to TNF-α.
TNF-α-responsive genes were selected based on LUTD patients’ NGS data and in silico analysis. SM and UE cells were treated with 10 ng/mL TNF-α and RNA isolated. Regulation of TNF-α-induced genes was studied by quantitative real-time polymerase chain reaction (qRT-PCR) and NGS. nCounter® miRNA Expression Assays (NanoString, Washington, Seattle, USA) were used to profile miRNAs. A cell proliferation assay was performed to evaluate the proliferative effects of TNF-α. To determine whether NFκB signalling was affected by the altered miRNAs, NFkB-luciferase reporter assays were performed.
After TNF-α treatment, NFκB2, RelB, and TNFAIP3 showed a progressive time and concentration-dependent upregulation, and responses were stronger in SM cells compared to UE cells. TNF-α treatment increased cell proliferation. miRNA expression profiling identified 17 miRNAs altered in both SM 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 SM cells, but downregulated in BOO. There was a cell-type-dependent difference in miRNA profiles, with SM cell-specific miRNAs downregulated after TNF-α treatment, in accordance with the downregulation of SMmarkers and loss of contractility in human patients. Transcriptome analysis of TNF-α treated cells was carried out and expression levels of predicted targets of disease-relevant miRNAs were identified. Ectopic over-expression of miR-199a-5p caused the downregulation of NFκB signalling in TNF-α-treated cells.
Our results confirm the important role of TNF-α in the regulation of BOO-specific miRNAs and identify miRNAs linking TNF-α signalling and fibrosis. Based on the reduced NFκB activation in miR-199a-5p-overexpressing SM cells and HEK293 cells, NFκB signalling may be directly affected by hsa-miR-199a-5p, which targets specific pathway elements. Compensatory upregulation of miRNAs inhibited by TNF-α might prevent organ remodelling and lead to novel therapeutic approaches for BOO-induced LUTD.