Ali Hashemi Gheinani

miR-199a-5p Regulates Urothelial Permeability and May Play a Role in Bladder Pain Syndrome

Defects in urothelial integrity resulting in leakage and activation of underlying sensory nerves are potential causative factors of bladder pain syndrome, a clinical syndrome of pelvic pain and urinary urgency/frequency in the absence of a specific cause. Herein, we identified the microRNA miR-199a-5p as an important regulator of intercellular junctions. On overexpression in urothelial cells, it impairs correct tight junction formation and leads to increased permeability. miR-199a-5p directly targets mRNAs encoding LIN7C, ARHGAP12, PALS1, RND1, and PVRL1 and attenuates their expression levels to a similar extent. Using laser microdissection, we showed that miR-199a-5p is predominantly expressed in bladder smooth muscle but that it is also detected in mature bladder urothelium and primary urothelial cultures. In the urothelium, its expression can be up-regulated after activation of cAMP signaling pathways. While validating miR-199a-5p targets, we delineated novel functions of LIN7C and ARHGAP12 in urothelial integrity and confirmed the essential role of PALS1 in establishing and maintaining urothelial polarity and junction assembly. The present results point to a possible link between miR-199a-5p expression and the control of urothelial permeability in bladder pain syndrome. Up-regulation of miR-199a-5p and concomitant down-regulation of its multiple targets might be detrimental to the establishment of a tight urothelial barrier, leading to chronic pain.

Deciphering microRNA code in pain and inflammation: lessons from bladder pain syndrome.

MicroRNAs (miRNAs), a novel class of molecules regulating gene expression, have been hailed as modulators of many biological processes and disease states. Recent studies demonstrated an important role of miRNAs in the processes of inflammation and cancer, however, there are little data implicating miRNAs in peripheral pain. Bladder pain syndrome/interstitial cystitis (BPS/IC) is a clinical syndrome of pelvic pain and urinary urgency/frequency in the absence of a specific cause. BPS is a chronic inflammatory condition that might share some of the pathogenetic mechanisms with its common co-morbidities inflammatory bowel disease (IBD), asthma and autoimmune diseases. Using miRNA profiling in BPS and the information about validated miRNA targets, we delineated the signaling pathways activated in this and other inflammatory pain disorders. This review projects the miRNA profiling and functional data originating from the research in bladder cancer and immune-mediated diseases on the BPS-specific miRNAs with the aim to gain new insight into the pathogenesis of this enigmatic disorder, and highlighting the common regulatory mechanisms of pain and inflammation.

MicroRNA MiR-199a-5p regulates smooth muscle cell proliferation and morphology by targeting WNT2 signaling pathway.

Background: MicroRNA miR-199a-5p, implicated in cell motility and proliferation, is highly expressed in bladder smooth muscle. Results: MiR-199a-5p regulates WNT, cytoskeleton, and cell cycle pathways in urothelial and smooth muscle cells and promotes myocardin-driven gene expression. Conclusion: MiR-199a-5p acts via its target WNT2 to control smooth muscle proliferation and morphology. Significance: MiR-199a-5p is a key modulator of smooth muscle hypertrophy, relevant for bladder organ remodeling. MicroRNA miR-199a-5p impairs tight junction formation, leading to increased urothelial permeability in bladder pain syndrome. Now, using transcriptome analysis in urothelial TEU-2 cells, we implicate it in the regulation of cell cycle, cytoskeleton remodeling, TGF, and WNT signaling pathways. MiR-199a-5p is highly expressed in the smooth muscle layer of the bladder, and we altered its levels in bladder smooth muscle cells (SMCs) to validate the pathway analysis. Inhibition of miR-199a-5p with antimiR increased SMC proliferation, reduced cell size, and up-regulated miR-199a-5p targets, including WNT2. Overexpression of WNT2 protein or treating SMCs with recombinant WNT2 closely mimicked the miR-199a-5p inhibition, whereas down-regulation of WNT2 in antimiR-expressing SMCs with shRNA restored cell phenotype and proliferation rates. Overexpression of miR-199a-5p in the bladder SMCs significantly increased cell size and up-regulated SM22, SM α-actin, and SM myosin heavy chain mRNA and protein levels. These changes as well as increased expression of ACTG2, TGFB1I1, and CDKN1A were mediated by up-regulation of the smooth muscle-specific transcriptional activator myocardin at mRNA and protein levels. Myocardin-related transcription factor A downstream targets Id3 and MYL9 were also induced. Up-regulation of myocardin was accompanied by down-regulation of WNT-dependent inhibitory Krüppel-like transcription factor 4 in miR-199a-5p-overexpressing cells. In contrast, Krüppel-like transcription factor 4 was induced in antimiR-expressing cells following the activation of WNT2 signaling, leading to repression of myocardin-dependent genes. MiR-199a-5p plays a critical role in the WNT2-mediated regulation of proliferative and differentiation processes in the smooth muscle and may behave as a key modulator of smooth muscle hypertrophy, which is relevant for organ remodeling.

Improved isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery

Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RNAs can be packaged in secreted urinary extracellular vesicles (uEVs) and thus protected from degradation. Urinary exosome preparations might contain specific miRNAs, relevant as biomarkers in renal and bladder diseases. Major difficulties in application of uEVs into the clinical environment are the high variability and low reproducibility of uEV isolation methods. Here we used five different methods to isolate uEVs and compared the size distribution, morphology, yield, presence of exosomal protein markers and RNA content of uEVs. We present an optimized ultracentrifugation and size exclusion chromatography approach for highly reproducible isolation for 50–150 nm uEVs, corresponding to the exosomes, from 50 ml urine. We profiled the miRNA content of uEVs and total urine from the same samples with the NanoString platform and validated the data using qPCR. Our results indicate that 18 miRNAs, robustly detected in uEVs were always present in the total urine. However, 15 miRNAs could be detected only in the total urine preparations and might represent naked circulating miRNA species. This is a novel unbiased and reproducible strategy for uEVs isolation, content normalization and miRNA cargo analysis, suitable for biomarker discovery studies.

miR-19b enhances proliferation and apoptosis resistance via the EGFR signaling pathway by targeting PP2A and BIM in non-small cell lung cancer

BackgroundEpidermal growth factor receptor (EGFR) mutations enable constitutive active downstream signaling of PI3K/AKT, KRAS/ERK and JAK/STAT pathways, and promote tumor progression by inducing uncontrolled proliferation, evasion of apoptosis and migration of non-small cell lung cancer (NSCLC). In addition, such EGFR mutations increase the susceptibility of patients with NSCLC to tyrosine kinase inhibitor (TKI) therapy, but treated patients will invariably relapse with resistant disease. A global understanding of underlying molecular mechanisms of EGFR signaling may improve the management of NSCLC patients.Methodsmicroarray analysis was performed to identify PI3K/AKT-regulated miRNAs. Phosphoproteomic analysis and cell based assays were performed using NSCLC cell lines lentivirally transduced with anti-miR or miR overexpressing constructs.ResultsHere, we show that 17 miRNAs including members of the miR-17~ 92 cluster are dysregulated following PI3K/AKT inhibition of EGFR mutant NSCLC cells. Bioinformatics analysis revealed that dysregulated miRNAs act in a concerted manner to enhance the activity of the EGFR signaling pathway. These findings were closely mirrored by attenuation of miR-17~ 92 family member miR-19b in NSCLC cell lines which resulted in reduced phosphorylation of ERK, AKT and STAT and effector proteins in EGFR mutant NSCLC cells. Consistent with this finding, cell cycle progression, clonogenic growth and migration were reduced and apoptosis was enhanced. Co-treatment of NSCLC cells with the tyrosine kinase inhibitor (TKI) gefitinib and anti-miR-19b construct reduced migration and clonogenic growth in a synergistic manner suggesting that EGFR and miR-19b act together to control oncogenic processes. Serine/threonine phosphatase PP2A subunit PPP2R5E and BCL2L11 encoding BIM were identified as major targets of miR-19b by target validation assays. Consistent with this finding, PP2A activity was strongly enhanced in NSCLC transduced with anti-miR-19b construct, but not in cells co-transduced with anti-miR-19b and shPPP2R5E, suggesting that PPP2R5E is a major constituent of the PP2A complex. Accordingly, enhanced proliferation by miR-19b was due to targeting PPP2R5E. In contrast, apoptosis resistance was mainly due to targeting BCL2L11.ConclusionOur results provide insight into the importance of targeting PPP2R5E and BCL2L11 by miR-19b in oncogenic processes of NSCLC. Attenuation of miR-19b expression could potentially be exploited in adjuvant therapy of EGFR mutant NSCLC.

Tumor Necrosis Factor-α Initiates miRNA-mRNA Signaling Cascades in Obstruction-Induced Bladder Dysfunction.

Bladder outlet obstruction (BOO) and the ensuing clinical lower urinary tract dysfunction are common in elderly patients. BOO is accompanied by urodynamic changes in bladder function and leads to organ fibrosis and ultimately loss of contractility. Comprehensive transcriptome analysis of bladder samples from human patients with different urodynamically defined phenotypes of BOO revealed tumor necrosis factor (TNF)-α as the top upstream signaling pathway regulator. Herein, we validated next-generation sequencing and pathway analysis in cell-based models using bladder smooth muscle and urothelial cells exposed to TNF-α. miRNA profiling and transcriptome analysis of TNF-α-treated bladder smooth muscle cells revealed striking similarities with human BOO. Using a comparative approach, TNF-specific and TNF-independent pathways were delineated in human biopsy specimens. Concomitant down-regulation of smooth muscle cell-specific miRNAs and smooth muscle markers after TNF-α treatment was in accordance with the loss of contractility in humans in advanced obstruction-induced bladder remodeling. The expression levels of four abundant TNF-regulated miRNAs were modulated; the compensatory up-regulation of miR-199a-5p reduced NF-κB signaling. Essential hubs of TNF-α signaling pathways mitogen-activated protein kinase kinase kinase (apoptosis signal-regulating kinase 1) and inhibitor of nuclear factor κ B kinase subunit β (IκB kinase β) were targeted by miR-199a-5p. miR-199a-5p might be part of a negative feedback loop, reducing the impact of TNF, whereas its down-regulation in acontractile bladders from BOO patients advances the disease. The compensatory up-regulation of miR-199a-5p together with TNF-α inhibition may be therapeutically beneficial.

Deletion of neuropilin 2 enhances detrusor contractility following bladder outlet obstruction

Chronic urethral obstruction and the ensuing bladder wall remodeling can lead to diminished bladder smooth muscle (BSM) contractility and debilitating lower urinary tract symptoms. No effective pharmacotherapy exists to restore BSM contractile function. Neuropilin 2 (Nrp2) is a transmembrane protein that is highly expressed in BSM. Nrp2 deletion in mice leads to increased BSM contraction. We determined whether genetic ablation of Nrp2 could restore BSM contractility following obstruction. Partial bladder outlet obstruction (pBOO) was created by urethral occlusion in mice with either constitutive and ubiquitous, or inducible smooth muscle-specific deletion of Nrp2, and Nrp2-intact littermates. Mice without obstruction served as additional controls. Contractility was measured by isometric tension testing. Nrp2 deletion prior to pBOO increased force generation in BSM 4 weeks following surgery. Deletion of Nrp2 in mice already subjected to pBOO for 4 weeks showed increased contractility of tissues tested 6 weeks after surgery compared with nondeleted controls. Assessment of tissues from patients with urodynamically defined bladder outlet obstruction revealed reduced NRP2 levels in obstructed bladders with compensated compared with decompensated function, relative to asymptomatic controls. We conclude that downregulation of Nrp2 promotes BSM force generation. Neuropilin 2 may represent a novel target to restore contractility following obstruction.

MP38-11 ROLE OF MICRORNAS, INHIBITED BY TNF-?, IN SMOOTH MUSCLE REMODELING DURING OUTLET OBSTRUCTION-INDUCED LOWER URINARY TRACT DYSFUNCTION

Interestingly, cystometric recordings from TREK-1 KO mice revealed a significant increase in the duration of inter-micturition interval, also expressed enhanced bladder capacity and increased number of nonvoiding contractions in comparison to WT mice. CONCLUSIONS: Our data suggest that the knockout of TREK1 channels has dual effects on detrusor contractility and micturition patterns, most likely due to expression of TREK-1 channels on both smooth muscle and neuronal cells.

MP82-08 THE POTENTIAL OF 2 MICRORNA CLUSTERS IN ELUCIDATION OF BIOLOGICAL FUNCTIONS OF SIGNALLING PATHWAYS REGULATED BY MICRORNAS IN UNDERACTIVE BLADDER

INTRODUCTION AND OBJECTIVES: MicroRNAs regulate diverse biological processes. Previously we identified miRNA-regulated pathways in bladder outlet obstruction (BOO)-induced bladder dysfunction. MiRNAs in a cluster reside in genomic proximity (<10 kb) and their expression might be mediated by common transcription factors. Here we probed functional associations of BOO phenotype-specific miRNAs and identified several co-expressed miRNA sub-networks. METHODS: MiRNA sequences and genomic coordinates were extracted from miRBase version 21. Large scale chromosomal mapping of human miRNA structural clusters was done using MIReStruC-1.0 package. Next-generation sequencing datasets of patients0 biopsies with urodynamically established BOO with and without detrusor overactivity (DO and BO groups, respectively) or with detrusor underactivity (UA group) were used to perform miRNA-mRNA integrated analysis and target pairing. Sequences were aligned with MAFFT version 7 and Clustal X 2.1 and manually refined with RALEE-RNA version 0.8. RESULTS: In DO group hsa-miR-376c-3p/hsa-miR-409-3p cluster was identified on chromosome 14. In BO group hsa-miR-889-3p/ hsa-miR-410-3p/hsa-miR-409-3p cluster was detected on chromosome 14. Three miRNA clusters were detected in UA group: hsa-miR-25-3p/ hsa-miR-106b-3p cluster on chromosome 7, and 2 clusters on chromosome 1: hsa-miR-199a-3p/hsa-miR-3120-3p cluster and hsa-miR429/hsa-miR-200b-3p cluster belonging to miR-200bc/429/548a family. Integrated miRNA-mRNA expression profiling revealed no significant target overlap, and 90% of targets of up-regulated miRNA clusters were also up-regulated. No major contribution of the miRNA clusters to the biological functions of miRNA-regulated pathways was detected in DO and BO groups. In UA group 2 down-regulated hsa-miR-199a-3p/hsamiR-3120-3p and hsa-miR-429/hsa-miR-200b-3p miRNA clusters were necessary and sufficient to determine the functions of all miRNAregulated pathways and their targets constituted the majority of miRNAregulated pathway elements. CONCLUSIONS: Multiple co-expressed miRNAs may cooperatively influence biological processes and the acontractile urodynamic phenotype in the underactive bladder. Elucidating the down-regulation mechanisms of these miRNA clusters may help determine the »point of no return« for the loss of bladder function during BOO.

MP82-19 VALIDATION OF TNF-? AS THE TOP UPSTREAM REGULATOR OF BLADDER REMODELING DURING OUTLET OBSTRUCTION-INDUCED LOWER URINARY TRACT DYSFUNCTION

INTRODUCTION AND OBJECTIVES: Clinically, mid-urethral sling procedure has become a regular and popular treatment for stress urinary incontinence (SUI). However, it has limited improvement if intrinsic sphincter deficiency dominantly and/or co-existing serious urgency symptom. We applied a new neuro-modulative sling in an acute urinary incontinence rat model to test if it can improve the urinary incontinence and urinary urgency simultaneously. METHODS: The neuro-modulative sling made by sling mesh along with small silver wire electrodes which were connected to electrical stimulator with the free ends in the middle of sling for electrical stimulation. An acute mixed urinary incontinence animal model was made by bilaterally pudendual nerve transaction (PNT, n1⁄421) or sham PNT (n1⁄420) followed by potassium chloride (4M, KCl) or saline (control, 0.9% NaCl) bladder perfusion. Urodynamic testing performed to confirm the changing of the bladder contractions. Leak point pressure (LPP) was also tested during filling cytometry. RESULTS: With current above 10mA and frequency above 50Hz result in significantly increased LPP during electrical stimulation delivering to the neuro-modulative sling (p<0.05). The continuous electrical stimulation with bi-polar square wave at the parameters of frequency to 5 Hz and pulse duration to 150 ms may decrease the increased bladder contractions significantly after PNT and KCl perfusion (p<0.01). CONCLUSIONS: The study demonstrated the neuro-modulative sling with different electrical stimulation parameters may improve mixed urinary incontinence in an acute animal model. Our future direction is studying its effects on a long-term animal model and possible complications if long-term implantation and use.