Benign prostatic obstruction (BPO) and associated lower urinary tract symptoms markedly diminish the quality of life in elderly men. BPO triggers chronic inflammation, leading to bladder dysfunction and a progressive decline in detrusor contractility. Previous next-generation sequencing (NGS) studies revealed altered RNA profiles, notably the significant downregulation of miR-424-5p, alongside TNF-alpha emerging as a key upstream regulator in BPO-induced bladder changes. In light of these findings, we hypothesize that targeting the TNF-alpha regulator could potentially restore miR-424-5p expression, offering therapeutic benefits in alleviating BPO-associated adverse effects. Our primary aim is to elucidate the targets of miR-424-5p, particularly its involvement in anti-inflammatory and anti-proliferative pathways. Additionally, we aim to dissect the mechanisms whereby TNF-alpha pathway and miR-424-5p orchestrate the changes in bladder wall after BPO.

Primary smooth muscle, CCD1 fibroblast and urothelial cells were treated with TNF-alpha, RNA isolated, and levels of pri-miR-424, ERG1 and NFKBIA determined by QPCR. NFkB-luciferase assays were performed to determine the effect of miRNAs on TNF-alpha signalling. 3’ non-coding and miRNA-binding regions of NFKB1, PRKCD, FGFR and PIKCD were cloned into pmirGLO dual-luciferase vector and target binding assay performed in cells, transfected with pre-miRNAs miR-424 and miR-199a. Pri-miR-424 promoter region and its parts containing predicted transcription start sequences was cloned into pBV-Luc vector and promoter activity determined by luciferase assay. CpG-rich part of the main promoter was deleted in CCD1 cells using CRISPR-Cas. Inhibitors of TNF signalling were used to determine the mechanisms of pri-miR424 down-regulation.

Compensatory overexpression of TNF alpha-inhibited miRNAs miR-199a-5p, miR-424-5p, miR-27b and miR-149-5p significantly reduced NFkB-driven luciferase gene expression.  Using pmirGLO assays we showed that miR-424-5p directly targets and down-regulates FGFR1, NFKB1, PRKCD and MAP2K1, important components of TNF signalling. Molecular dissection of the pri-miR-424 promoter region demonstrated a CpG-rich area with a strong transcriptional activity. Deletion of this part of the promoter reduced the expression of pri-miR-424 to 10%, but did not abolish the TNF-mediated inhibition of its synthesis, indicating that the first 1100 bp of its promoter contained the regulatory sequences. JSH-23, a specific inhibitor of NFkB p65 translocation, abrogated TNF-alpha mediated down-regulation of pri-miR-424 and EGR1, without affecting up-regulation of NFKBIA. We show that CEBPB transcription factor is involved in miR-424 down-regulation together with p65.

Anti-inflammatory miR-424-5p inhibits TNF alpha by targeting and down-regulating important hubs of NFkB signalling. In turn, NF-kappa B p65 in association with C/EBP beta bind and down-regulate the expression of miR-424. The promoter region of pri-miR-424 has several regions, including CpG-rich areas, with high transcriptional activity, however, the regulatory region of first 1100 bp harbours the binding sites of CEBPB, important for TNF-mediated down-regulation. JSH-23 and similar inhibitors could be useful tools to sustain miR-424 levels and reduce inflammation.

Down-regulated miRNAs have a high impact on TNF alpha signalling. The importance of the targets of down-regulated miR-424-5p attenuating NFkB-mediated inflammation indicates that finding the pharmacological ways to enhance its expression might have therapeutic benefits for preserving bladder contractility during BPO.

Continence 12S (2024) 101598
DOI: 10.1016/j.cont.2024.101598