Increased expression of CXCL6 in secretory cells drives fibroblast collagen synthesis and is associated with increased mortality in idiopathic pulmonary fibrosis.

RATIONALE: Recent data suggest that the localisation of airway epithelial cells in the distal lung in idiopathic pulmonary fibrosis (IPF) may drive pathology. We set out to discover whether chemokines expressed in these ectopic airway epithelial cells may contribute to the pathogenesis of IPF. METHODS: We analysed whole lung and single-cell transcriptomic data obtained from patients with IPF. In addition, we measured chemokine levels in blood, bronchoalveolar lavage (BAL) of IPF patients and air-liquid interface cultures. We employed ex vivo donor and IPF lung fibroblasts and an animal model of pulmonary fibrosis to test the effects of chemokine signalling on fibroblast function. RESULTS: By analysis of whole-lung transcriptomics, protein and BAL, we discovered that CXCL6 (a member of the interleukin-8 family) was increased in patients with IPF. Elevated CXCL6 levels in the BAL of two cohorts of patients with IPF were associated with poor survival (hazard ratio of death or progression 1.89, 95% CI 1.16-3.08; n=179, p=0.01). By immunostaining and single-cell RNA sequencing, CXCL6 was detected in secretory cells. Administration of mCXCL5 (LIX, murine CXCL6 homologue) to mice increased collagen synthesis with and without bleomycin. CXCL6 increased collagen I levels in donor and IPF fibroblasts 4.4-fold and 1.7-fold, respectively. Both silencing of and chemical inhibition of CXCR1/2 blocked the effects of CXCL6 on collagen, while overexpression of CXCR2 increased collagen I levels 4.5-fold in IPF fibroblasts. CONCLUSIONS: CXCL6 is expressed in ectopic airway epithelial cells. Elevated levels of CXCL6 are associated with IPF mortality. CXCL6-driven collagen synthesis represents a functional consequence of ectopic localisation of airway epithelial cells in IPF.

End-Stage Idiopathic Pulmonary Fibrosis Lung Microenvironment Promotes Impaired NK Activity.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic age-related chronic lung disease characterized by the accumulation of senescent cells. Whether impaired immune response is responsible for the accumulation of senescent cells in the IPF lung remains unknown. In this study, we characterized the NK phenotype in IPF lungs via flow cytometry using 5-dodecanoylaminofluorescein di-ß-d-galactopyranoside, markers of tissue residence, and chemokine receptors. The effect of the lung microenvironment was evaluated using lung fibroblast (LF) conditioned media (CM), and the bleomycin-induced pulmonary fibrosis mouse model was used to assess the in vivo relationship between NK cells and the accumulation of senescent cells. We found that NK cells from the lower lobe of IPF patients exhibited immune-senescent and impaired CD57-NKG2A+ phenotype. We also observed that culture of NK cells from healthy donors in CM from IPF lower lobe lung fibroblasts induced a senescent-like phenotype and impaired cytotoxic capacity. There is an impaired NK recruitment by LF, and NKs presented decreased migration toward their CM. In addition, NK cell-depleted mice treated with bleomycin showed increased collagen deposition and accumulation of different populations of senescent cells compared with controls. The IPF lung microenvironment induces a dysfunctional NK phenotype limiting the clearance of lung senescent cells and the resolution of lung fibrosis. We propose that impaired NK activity could be one of the mechanisms responsible for perpetuating the accumulation of senescent cells in IPF lungs.

TGF-ß1 Inhibition of ACE2 Mediated by miRNA Uncovers Novel Mechanism of SARS-CoV-2 Pathogenesis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for COVID-19, utilizes receptor binding domain (RBD) of spike glycoprotein to interact with angiotensin (Ang)-converting enzyme 2 (ACE2). Altering ACE2 levels may affect entry of SARS-CoV-2 and recovery from COVID-19. Decreased cell surface density of ACE2 leads to increased local levels of Ang II and may contribute to mortality resulting from acute lung injury and fibrosis during COVID-19. Studies published early during the COVID-19 pandemic reported that people with cystic fibrosis (PwCF) had milder symptoms, compared to people without CF. This finding was attributed to elevated ACE2 levels and/or treatment with the high efficiency CFTR modulators. Subsequent studies did not confirm these findings reporting variable effects of CFTR gene mutations on ACE2 levels. Transforming growth factor (TGF)-ß signaling is essential during SARS-CoV-2 infection and dominates the chronic immune response in severe COVID-19, leading to pulmonary fibrosis. TGF-ß1 is a gene modifier associated with more severe lung disease in PwCF but its effects on the COVID-19 course in PwCF is unknown. To understand whether TGF-ß1 affects ACE2 levels in the airway, we examined miRNAs and their gene targets affecting SARS-CoV-2 pathogenesis in response to TGF-ß1. Small RNAseq and micro(mi)RNA profiling identified pathways uniquely affected by TGF-ß1, including those associated with SARS-CoV-2 invasion, replication, and the host immune responses. TGF-ß1 inhibited ACE2 expression by miR-136-3p and miR-369-5p mediated mechanism in CF and non-CF bronchial epithelial cells. ACE2 levels were higher in two bronchial epithelial cell models expressing the most common CF-causing mutation in CFTR gene F508del, compared to controls without the mutation. After TGF-ß1 treatment, ACE2 protein levels were still higher in CF, compared to non-CF cells. TGF-ß1 prevented the modulator-mediated rescue of F508del-CFTR function while the modulators did not prevent the TGF-ß1 inhibition of ACE2 levels. Finally, TGF-ß1 reduced the interaction between ACE2 and the recombinant spike RBD by lowering ACE2 levels and its binding to RBD. Our data demonstrate novel mechanism whereby TGF-ß1 inhibition of ACE2 in CF and non-CF bronchial epithelial cells may modulate SARS-CoV-2 pathogenicity and COVID-19 severity. By reducing ACE2 levels, TGF-ß1 may decrease entry of SARS-CoV-2 into the host cells while hindering the recovery from COVID-19 due to loss of the anti-inflammatory and regenerative effects of ACE2. The above outcomes may be modulated by other, miRNA-mediated effects exerted by TGF-ß1 on the host immune responses, leading to a complex and yet incompletely understood circuitry.

Cell signaling and regulation of CFTR expression in cystic fibrosis cells in the era of high efficiency modulator therapy.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP- and protein kinase A (PKA)-regulated channel, expressed on the luminal surface of secretory and absorptive epithelial cells. CFTR has a complex, cell-specific regulatory network playing a major role in cAMP- and Ca2+-activated secretion of electrolytes. It secretes intracellular Cl- and bicarbonate and regulates absorption of electrolytes by differentially controlling the activity of the epithelial Na+ channel (ENaC) in colon, airways, and sweat ducts. The CFTR gene expression is regulated by cell-specific, time-dependent mechanisms reviewed elsewhere [1]. This review will focus on the transcriptional, post-transcriptional, and translational regulation of CFTR by cAMP-PKA, non-coding (nc)RNAs, and TGF-ß signaling pathways in cystic fibrosis (CF) cells.

Fatty acid nitroalkene reversal of established lung fibrosis.

Tissue fibrosis occurs in response to dysregulated metabolism, pro-inflammatory signaling and tissue repair reactions. For example, lungs exposed to environmental toxins, cancer therapies, chronic inflammation and other stimuli manifest a phenotypic shift to activated myofibroblasts and progressive and often irreversible lung tissue scarring. There are no therapies that stop or reverse fibrosis. The 2 FDA-approved anti-fibrotic drugs at best only slow the progression of fibrosis in humans. The present study was designed to test whether a small molecule electrophilic nitroalkene, nitro-oleic acid (NO2-OA), could reverse established pulmonary fibrosis induced by the intratracheal administration of bleomycin in C57BL/6 mice. After 14 d of bleomycin-induced fibrosis development in vivo, lungs were removed, sectioned and precision-cut lung slices (PCLS) from control and bleomycin-treated mice were cultured ex vivo for 4 d with either vehicle or NO2-OA (5 µM). Biochemical and morphological analyses showed that over a 4 d time frame, NO2-OA significantly inhibited pro-inflammatory mediator and growth factor expression and reversed key indices of fibrosis (hydroxyproline, collagen 1A1 and 3A1, fibronectin-1). Quantitative image analysis of PCLS immunohistology reinforced these observations, revealing that NO2-OA suppressed additional hallmarks of the fibrotic response, including alveolar epithelial cell loss, myofibroblast differentiation and proliferation, collagen and α-smooth muscle actin expression. NO2-OA also accelerated collagen degradation by resident macrophages. These effects occurred in the absence of the recognized NO2-OA modulation of circulating and migrating immune cell activation. Thus, small molecule nitroalkenes may be useful agents for reversing pathogenic fibrosis of lung and other organs.

Simultaneous Pharmacologic Inhibition of Yes-Associated Protein 1 and Glutaminase 1 via Inhaled Poly(Lactic-co-Glycolic) Acid-Encapsulated Microparticles Improves Pulmonary Hypertension.

Background Pulmonary hypertension (PH) is a deadly disease characterized by vascular stiffness and altered cellular metabolism. Current treatments focus on vasodilation and not other root causes of pathogenesis. Previously, it was demonstrated that glutamine metabolism, as catalyzed by GLS1 (glutaminase 1) activity, is mechanoactivated by matrix stiffening and the transcriptional coactivators YAP1 (yes-associated protein 1) and transcriptional coactivator with PDZ-binding motif (TAZ), resulting in pulmonary vascular proliferation and PH. Pharmacologic inhibition of YAP1 (by verteporfin) or glutaminase (by CB-839) improved PH in vivo. However, systemic delivery of these agents, particularly YAP1 inhibitors, may have adverse chronic effects. Furthermore, simultaneous use of pharmacologic blockers may offer additive or synergistic benefits. Therefore, a strategy that delivers these drugs in combination to local lung tissue, thus avoiding systemic toxicity and driving more robust improvement, was investigated. Methods and Results We used poly(lactic-co-glycolic) acid polymer-based microparticles for delivery of verteporfin and CB-839 simultaneously to the lungs of rats suffering from monocrotaline-induced PH. Microparticles released these drugs in a sustained fashion and delivered their payload in the lungs for 7 days. When given orotracheally to the rats weekly for 3 weeks, microparticles carrying this drug combination improved hemodynamic (right ventricular systolic pressure and right ventricle/left ventricle+septum mass ratio), histologic (vascular remodeling), and molecular markers (vascular proliferation and stiffening) of PH. Importantly, only the combination of drug delivery, but neither verteporfin nor CB-839 alone, displayed significant improvement across all indexes of PH. Conclusions Simultaneous, lung-specific, and controlled release of drugs targeting YAP1 and GLS1 improved PH in rats, addressing unmet needs for the treatment of this deadly disease.

Differential Gene Expression Analysis Reveals Global LMTK2 Regulatory Network and Its Role in TGF-ß1 Signaling.

Lemur tyrosine kinase 2 (LMTK2) is a transmembrane Ser/Thr kinase whose role has been increasingly recognized; however, when compared to other kinases, understanding of the LMTK2 networks and biological functions is still limited. Recent data have shown that transforming growth factor (TGF)-ß1 plays a role in modulating LMTK2 function by controlling its endocytic trafficking in human bronchial epithelial cells. Here, we aimed to unveil the LMTK2 regulatory network and elucidate how it affects cellular functions and disease pathways in either TGF-ß1 dependent or independent manner. To understand how the LMTK2 and TGF-ß1 pathways interconnect, we knocked down (KD) LMTK2 using small(si)RNA-mediated silencing in human bronchial epithelial CFBE41o- cells, treated cells with TGF-ß1 or vehicle control, and performed differential gene expression analysis by RNA sequencing (RNAseq). In vehicle-treated cells, LMTK2 KD affected expression of 2,506 genes while it affected 4,162 genes after TGF-ß1 stimulation. Bioinformatics analysis shows that LMTK2 is involved in diverse cellular functions and disease pathways, such as cell death and survival, cellular development, and cancer susceptibility. In summary, our study increases current knowledge about the LMTK2 network and its intersection with the TGF-ß1 signaling pathway. These findings will serve as basis for future exploration of the predicted LMTK2 interactions and signaling pathways.

Human Lung-Resident Macrophages Colocalize with and Provide Costimulation to PD1hi Tissue-Resident Memory T Cells.

Rationale: Tissue-resident memory T cells (TRM) play a critical role in the defense against inhaled pathogens. The isolation and study of human lung tissue-resident memory T cells and lung-resident macrophages (MLR) are limited by experimental constraints. Objectives: To characterize the spatial and functional relationship between MLR and human lung tissue-resident memory T cells using ex vivo lung perfusion (EVLP). Methods: TRM and MLR were isolated using EVLP and intraperfusate-labeled CD45 antibody. Cells isolated after 6 hours of EVLP were analyzed using spectral flow cytometry. Spatial relationships between CD3+ and CD68+ cells were explored with multiplexed immunohistochemistry. Functional relationships were determined by using coculture and T-cell-receptor complex signal transduction. Measurements and Main Results: Lungs from 8 research-consenting organ donors underwent EVLP for 6 hours. We show that human lung TRM and MLR colocalize within the human lung, preferentially around the airways. Furthermore, we found that human lung CD8+ TRM are composed of two functionally distinct populations on the basis of PD1 (programed cell death receptor 1) and ZNF683 (HOBIT) protein expression. We show that MLR provide costimulatory signaling to PD1hi CD4+ and CD8+ lung TRM,, augmenting the effector cytokine production and degranulation of TRM. Conclusions: EVLP provides an innovative technique to study resident immune populations in humans. Human MLR colocalize with and provide costimulation signaling to TRM, augmenting their effector function.

The Ago2-miRNA-co-IP Assay to Study TGF- ß1 Mediated Recruitment of miRNA to the RISC in CFBE Cells.

Micro(mi)RNAs are short, non-coding RNAs that mediate the RNA interference (RNAi) by post-transcriptional mechanisms. Specific miRNAs are recruited to the cytoplasmic RNA induced silencing complex (RISC). Argonaute2 (Ago2), an essential component of RISC, facilitates binding of miRNA to the target-site on mRNA, followed by cleaving the miRNA-mRNA duplex with its endonuclease activity. RNAi is mediated by a specific pool of miRNAs recruited to RISC, and thus is referred to as the functional pool. The cellular levels of many miRNAs are affected by the cytokine Transforming Growth Factor-ß1 (TGF-ß1). However, little is known about whether the TGF-ß1 affects the functional pools of these miRNAs. The Ago2-miRNA-co-IP assay, discussed in this manuscript, is designed to examine effects of TGF-ß1 on the recruitment of miRNAs to RISC and it helps to determine whether changes in the cellular miRNA levels correlate with changes in the RISC-associated, functional pools. The general principles of the assay are as follows. Cultured cells treated with TGF-ß1 or vehicle control are lysed and the endogenous Ago2 is immunoprecipitated with immobilized anti-Ago2 antibody, and the active miRNAs complexed with Ago2 are isolated with a RISC immunoprecipitation (RIP) assay kit. The miRNAs are identified with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) using miRNA-specific stem-looped primers during reverse transcription, followed by PCR using miRNA-specific forward and reverse primers, and TaqMan hydrolysis probes.

The Role of MicroRNA in the Airway Surface Liquid Homeostasis.

Mucociliary clearance, mediated by a coordinated function of cilia bathing in the airway surface liquid (ASL) on the surface of airway epithelium, protects the host from inhaled pathogens and is an essential component of the innate immunity. ASL is composed of the superficial mucus layer and the deeper periciliary liquid. Ion channels, transporters, and pumps coordinate the transcellular and paracellular movement of ions and water to maintain the ASL volume and mucus hydration. microRNA (miRNA) is a class of non-coding, short single-stranded RNA regulating gene expression by post-transcriptional mechanisms. miRNAs have been increasingly recognized as essential regulators of ion channels and transporters responsible for ASL homeostasis. miRNAs also influence the airway host defense. We summarize the most up-to-date information on the role of miRNAs in ASL homeostasis and host-pathogen interactions in the airway and discuss concepts for miRNA-directed therapy.

TGF-ß1 Augments the Apical Membrane Abundance of Lemur Tyrosine Kinase 2 to Inhibit CFTR-Mediated Chloride Transport in Human Bronchial Epithelia.

The most common disease-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, F508del, leads to cystic fibrosis (CF), by arresting CFTR processing and trafficking to the plasma membrane. The FDA-approved modulators partially restore CFTR function and slow down the progression of CF lung disease by increasing processing and delivery to the plasma membrane and improving activity of F508del-CFTR Cl- channels. However, the modulators do not correct compromised membrane stability of rescued F508del-CFTR. Transforming growth factor (TGF)-ß1 is a well-established gene modifier of CF associated with worse lung disease in F508del-homozygous patients, by inhibiting CFTR biogenesis and blocking the functional rescue of F508del-CFTR. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein localized at the apical and basolateral membrane domain of human bronchial epithelial cells. Phosphorylation of the apical membrane CFTR by LMTK2 triggers its endocytosis and reduces the abundance of membrane-associated CFTR, impairing the CFTR-mediated Cl- transport. We have previously shown that LMTK2 knockdown improves the pharmacologically rescued F508del-CFTR abundance and function. Thus, reducing the LMTK2 recruitment to the plasma membrane may provide a useful strategy to potentiate the pharmacological rescue of F508del-CFTR. Here, we elucidate the mechanism of LMTK2 recruitment to the apical plasma membrane in polarized CFBE41o- cells. TGF-ß1 increased LMTK2 abundance selectively at the apical membrane by accelerating its recycling in Rab11-positive vesicles without affecting LMTK2 mRNA levels, protein biosynthesis, or endocytosis. Our data suggest that controlling TGF-ß1 signaling may attenuate recruitment of LMTK2 to the apical membrane thereby improving stability of pharmacologically rescued F508del-CFTR.

NMR-derived targeted serum metabolic biomarkers appraisal of bladder cancer: A pre- and post-operative evaluation.

With high morbidity and mortality, urinary bladder cancer (BC) ranks fifth among common cancers globally. The inherent limitations of urine cytology and cystoscopy, and marginal enhancements in the rate of survival promt us to develop surrogate serum based metabolic biomarkers of screening, identification, and follow-up protocols of management for BC patients. Earlier, we exhibited that abnormal expression levels of dimethylamine (DMA), malonate, lactate, glutamine, histidine, and valine in serum may be used as signature metabolites to differentiate BC from healthy controls (HC) (J. Proteome Res. 2013; 12(12):5839-50). Here we further gauge and validate these observations by comparing pre-operative to post-operative follow-up BC patients. This study was conducted on 160 sera samples involving HC (n = 52), pre-operative (n = 55) and post-operative (n = 53) BC cases. 1H nuclear magnetic resonance (NMR) spectroscopy was used to generate serum metabolic profiles and to gauge aberrantly expressed metabolites. The targeted metabolomic approach revealed that the expression levels of these signature metabolites were progressively and significantly decreased in post-operative follow-up at the interval of 30, 60, and 90 days compared to pre-operative BC sera samples and were maintained at HC levels. Serum metabolic biomarkers appear to be an inspiring and least-invasive tactic for detection and prognosticating BC patient follow-up.

Precise Targeting of miRNA Sites Restores CFTR Activity in CF Bronchial Epithelial Cells.

MicroRNAs that are overexpressed in cystic fibrosis (CF) bronchial epithelial cells (BEC) negatively regulate CFTR and nullify the beneficial effects of CFTR modulators. We hypothesized that it is possible to reverse microRNA-mediated inhibition of CFTR using CFTR-specific target site blockers (TSBs) and to develop a drug-device combination inhalation therapy for CF. Lead microRNA expression was quantified in a series of human CF and non-CF samples and in vitro models. A panel of CFTR 3' untranslated region (UTR)-specific locked nucleic acid antisense oligonucleotide TSBs was assessed for their ability to increase CFTR expression. Their effects on CFTR activity alone or in combination with CFTR modulators were measured in CF BEC models. TSB encapsulation in poly-lactic-co-glycolic acid (PLGA) nanoparticles was assessed as a proof of principle of delivery into CF BECs. TSBs targeting the CFTR 3' UTR 298-305:miR-145-5p or 166-173:miR-223-3p sites increased CFTR expression and anion channel activity and enhanced the effects of ivacaftor/lumacaftor or ivacaftor/tezacaftor in CF BECs. Biocompatible PLGA-TSB nanoparticles promoted CFTR expression in primary BECs and retained desirable biophysical characteristics following nebulization. Alone or in combination with CFTR modulators, aerosolized CFTR-targeting TSBs encapsulated in PLGA nanoparticles could represent a promising drug-device combination therapy for the treatment for CFTR dysfunction in the lung.

Transforming Growth Factor-ß1 Selectively Recruits microRNAs to the RNA-Induced Silencing Complex and Degrades CFTR mRNA under Permissive Conditions in Human Bronchial Epithelial Cells.

Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene lead to cystic fibrosis (CF). The most common mutation F508del inhibits folding and processing of CFTR protein. FDA-approved correctors rescue the biosynthetic processing of F508del-CFTR protein, while potentiators improve the rescued CFTR channel function. Transforming growth factor (TGF-ß1), overexpressed in many CF patients, blocks corrector/potentiator rescue by inhibiting CFTR mRNA in vitro. Increased TGF-ß1 signaling and acquired CFTR dysfunction are present in other lung diseases. To study the mechanism of TGF-ß1 repression of CFTR, we used molecular, biochemical, and functional approaches in primary human bronchial epithelial cells from over 50 donors. TGF-ß1 destabilized CFTR mRNA in cells from lungs with chronic disease, including CF, and impaired F508del-CFTR rescue by new-generation correctors. TGF-ß1 increased the active pool of selected micro(mi)RNAs validated as CFTR inhibitors, recruiting them to the RNA-induced silencing complex (RISC). Expression of F508del-CFTR globally modulated TGF-ß1-induced changes in the miRNA landscape, creating a permissive environment required for degradation of F508del-CFTR mRNA. In conclusion, TGF-ß1 may impede the full benefit of corrector/potentiator therapy in CF patients. Studying miRNA recruitment to RISC under disease-specific conditions may help to better characterize the miRNAs utilized by TGF-ß1 to destabilize CFTR mRNA.

MicroRNA-21 could be a molecular marker to predict the recurrence of nonmuscle invasive bladder cancer.

INTRODUCTION: High relapse rate of nonmuscle invasive bladder cancer (NMIBC) is a major challenge. Overexpression of microRNA-21 (miR-21) which targets phosphatase and tensin homolog (PTEN), a gene associated with malignancy, has been reported in the bladder tumor tissue compared to normal mucosa by us and others. We have tested whether miR-21 levels in bladder mucosa could predict tumor recurrence. METHODS: In a prospective cohort setting, tumor tissues and normal bladder mucosa (NBM) were taken from BC patients during transurethral resection of bladder tumor. Age- and ethnicity-matched NBM from benign prostate hyperplasia patients was taken as controls. The expression of miR-21 was analyzed using quantitative reverse transcription polymerase chain reaction. Patients were followed for 4 years for tumor reoccurrence. Postoperative recurrence were recorded and calculated by Kaplan-Meier curve. RESULTS: In 31 patients, miR-21 was up-regulated (>4-fold, P = 0.003), and PTEN levels were significantly lower (<7-folds, P = 0.001) in tumor tissue relative to NBM. Moreover, the fold change in miR-21 levels was significantly higher (>3-folds, P = 0.03) in patients showing recurrence compared to those in which tumor did not recur. Further, Kaplan-Meier analysis shows overexpression of miR-21 corresponds to less time to recurrence with higher cumulative hazard. CONCLUSION: We found overexpression of miR-21 in tumor tissue and its association with recurrence, time to recurrence and invasiveness in BC. Quantification of miR-21 along with other pathological parameters could be more objective molecular approach to predict recurrence in NMIBC.

Bladder cancer: Micro RNAs as biomolecules for prognostication and surveillance.

INTRODUCTION: Bladder cancer (BC) has varied clinical behavior in terms of recurrence and progression. Current pathological characteristics are insufficient to prognosticate the outcome of a given treatment. Cellular metabolic regulatory molecules, such as micro RNA (miRNA), could be a potential biomarker to prognosticate the treatment outcomes. MATERIALS AND METHODS: PubMed and Google Scholar databases were searched for publications from 1990 to 2016, related to miRNA biogenesis, its function, and role in the pathogenesis of bladder as well as other cancers. Articles were searched using MeSH terms micrornas, micrornas AND neoplasm, and micrornas AND urinary bladder neoplasm. Out of the 108 publications reviewed 75 references were selected based on the clinical relevance. Articles were reviewed to assess the role of miRNA in various cancers and those in BC as a diagnostic or therapeutic tool. RESULTS: More than 35 miRNAs were found to be associated with different pathways of cellular dedifferentiation, proliferation, and progression of BC as well as other cancers. A normal looking mucosa may show molecular changes preceding phenotypic changes in the form of varied expression of miR-129, miR-200a, and miR-205. miR-214, miR-99a, and miR-125b have been shown to be potential urinary biomarkers of BC. miRNAs could act as a repressor for protein molecule functioning or activator of different pathways to be used as a therapeutic target too. CONCLUSIONS: Despite certain limitations, such as instability, rapid plasma clearance, and targeting antagonist proteins of cellular metabolic pathways, miRNAs have potential to be studied as a biomarker or a therapeutic target for BC.

Predictors for progression of metastatic prostate cancer to castration-resistant prostate cancer in Indians.

BACKGROUND & OBJECTIVES: There is lack of data on natural history and progression of prostate cancer (PC) which have implications in the management of the disease. We undertook this retrospective study to analyze factors predicting progression of metastatic PC to castration-resistant prostate cancer (CRPC) in Indian men. METHODS: Complete records of 223 of the 489 patients with metastatic PC were obtained from computerized data based system in a tertiary care hospital in north India between January 2000 to June 2012. Patients with follow up of < 6 months were excluded. Age (≤ and > 65 yr), baseline PSA (< and ≥ 50 ng/ml), bone scan and Gleason score (≤7 and >7) were recorded. Extent of bone disease (EOD) was stratified according to the number of bone lesions i.e., < 5, 5-10, > 10. CRPC was defined as two consecutive PSA rise of > 50 per cent from nadir or an absolute value of > 5 ng/ml. RESULTS: Mean age of patients was 61.5 ± 12.45 yr and their PSA level was 325.6 ± 631.35 ng/dl. Of the 223 patients, 193 (86%) progressed to CRPC at median time of 10.7 (4-124) months. Median follow up was 24 (6-137) months. On univariate and multivariate analyses EOD on bone scan was found to be a significant predictor ( P=0.006) for time to CRPC. Median time to CRPC was 10 months (CI 95%, 7.5-12.48) with >10 lesions or super scan versus 16 months (CI 95%, 10.3-21.6) with <10 bone lesion (P=0.01). Ninety (46.6 %) patients of CRPC died with median time to death from time of CRPC 21 (10-120) months. INTERPRETATION & CONCLUSIONS: Median time for progression of metastatic PC to CRPC ranged from 10-16 months depending on the extent of the bone involvement. In Indians, the aggressive course of advanced prostate cancer warrants further clinical trials to explore the need for additional treatment along with initial castration.

Molecular cystoscopy: Micro-RNAs could be a marker for identifying genotypic changes for transitional cell carcinoma of the urinary bladder.

INTRODUCTION: Normal-looking mucosa may harbor genetic changes preceding a visible tumor. This study was aimed at exploring the role of the quantitative expression of micro-RNAs (miRNAs) in bladder cancer tissue in comparison with normal mucosa and healthy controls (HCs) as a molecular marker. MATERIALS AND METHODS: Between October 2011 to December 2012, tissue from the bladder tumor of 21 patients (cases tumor, CT), normal mucosa (case control, CC) of the same patients (n-21) and normal bladder mucosa from 10 HCs were obtained. miRNAs of angiogenesis, endothelial mesenchymal transition and apoptosis were quantified using stem-loop RT Taq Man polymerase chain reaction. Statistical analysis was performed using the Chi square and independent sample T tests by using SPSS version 16. RESULTS: The mean age of the patients and controls were 55.41 ± 11.03 and 52.14 ± 13.04 years. miR-21, miR-205, miR-126, miR-10b and miR-200a were highly expressed in CT (P < 0.027, <0.048, <0.025, <0.029 and < 0.005) as compared with HC. Expression of miR-21 and miR-129 were both correlated with grade and stage (P = 0.001 and < 0.009, respectively) and the level of expression was different in the same grade of non-muscle invasive tumors. The fold change of miR129, miR205 and miR200a was significantly higher in the normal-looking mucosa of bladder tumor patients than the HC (P < 0.005). CONCLUSION: Expression of miR129, miR205 and miR200a in the normal-looking mucosa of bladder cancer patients was significantly higher than the normal mucosa of a HC. This may help in predicting recurrence and formulating the follow-up strategy.

Low- and high-grade bladder cancer determination via human serum-based metabolomics approach.

To address the shortcomings of urine cytology and cystoscopy for probing and grading urinary bladder cancer (BC), we applied (1)H nuclear magnetic resonance (NMR) spectroscopy as a surrogate method for the identification of BC. This study includes 99 serum samples comprising low-grade (LG; n = 36) and high-grade (HG; n = 31) BC as well as healthy controls (HC; n = 32). (1)H NMR-derived serum data were analyzed using orthogonal partial least-squares discriminant analysis (OPLS-DA). OPLS-DA-derived model validity was confirmed using an internal and external cross-validation. Internal validation was performed using the initial samples (n = 99) data set. External validation was performed on a new batch of suspected BC patients (n = 106) through a double-blind study. Receiver operating characteristic (ROC) curve analysis was also performed. OPLS-DA-derived serum metabolomics (six biomarkers, ROC; 0.99) were able to discriminate 95% of BC cases with 96% sensitivity and 94% specificity when compared to HC. Likewise (three biomarkers, ROC; 0.99), 98% of cases of LG were able to differentiate from HG with 97% sensitivity and 99% specificity. External validation reveals comparable results to the internal validation. (1)H NMR-based serum metabolic screening appears to be a promising and less invasive approach for probing and grading BC in contrast to the highly invasive and painful cystoscopic approach for BC detection.