Abnormal Bladder Epithelial Cell Gene Expression in BPS/IC

BPS/IC 中膀胱上皮细胞基因表达异常

• 批准号: 8597942
• 负责人: Susan F. Keay
• 金额: --
• 依托单位: BALTIMORE VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-10-01 至 2016-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8597942
• 关键词: ATP Receptors; Abnormal Cell; Address; Affect; American; Biopsy; Biopsy Specimen; Bladder; CBA/J Mouse; Cell Culture Techniques; Cell Line; Cell Proliferation; Cells; Chromatin; Chronic; Clinical Trials; Complement; Complex; Coupled; DTR gene; Data; Development; Diagnosis; Disease; E-Cadherin; Epigenetic Process; Epithelial; Epithelial Cells; Epithelium; Etiology; Frequencies; Gene Expression; Gene Expression Regulation; Genes; Glycopeptides; Goals; Growth Factor; HDAC1 gene; HDAC2 gene; Healthcare; Healthcare Systems; Histone Acetylation; Histones; Human; In Vitro; Increased frequency of micturition; Interstitial Cystitis; Laboratories; Lead; MMP2 gene; Mechanics; Metalloproteases; MicroRNAs; Modeling; National Institute of Diabetes and Digestive and Kidney Diseases; Neurotransmitters; Normal Cell; Nuclear; Pathogenesis; Patients; Permeability; Phenotype; Play; Polymerase Chain Reaction; Population; Production; Proteins; Proteoglycan; Regulation; Regulator Genes; Reporting; Research; Role; Sampling; Small Interfering RNA; Staging; Stretching; Symptoms; Syndrome; Techniques; Testing; Theophylline; Tight Junctions; Time; Toxin; Transcriptional Regulation; Transfection; UPK3 gene; Ulcer; United States; Up-Regulation; Urine; Urologic Diseases; Veterans; Vimentin; Western Blotting; alanylalanine; base; bladder pain; chromatin immunoprecipitation; chromatin remodeling; effective therapy; epithelial to mesenchymal transition; experience; gain of function; genetic regulatory protein; histone modification; in vivo; inhibitor/antagonist; micturition urgency; monolayer; occludin; prolylvaline; protein expression; public health relevance; receptor; repaired; research study; response; transcription factor; valyl-valyl-valine

DESCRIPTION (provided by applicant): Bladder pain syndrome/interstitial cystitis (BPS/IC) is a chronic painful bladder syndrome that affects the lives of approximately one million people in the United States. BPS/IC is also becoming increasingly important for the veterans population as it was reported in 2006 to be the fourth most common urologic disease as well as the primary diagnosis for 1.4% of all VA healthcare users (rate increased 13.7% and raw numbers increased 38% from 1999-2002, most recent data available). The etiology of BPS/IC is unknown, and there is currently no reliably effective treatment. It therefore continues to be important to study the pathogenesis of this debilitating disorder, in order to systematically devise effective, disease-specific therapies. Epithelial abnormalities are the most consistent findings in bladder biopsies from BPS/IC patients, including denudation, tears, or thinning of the epithelium, coupled with abnormal proteoglycan and protein expression (including tight junction and adherens proteins, vimentin, and uroplakin III). Explanted BPS/IC bladder epithelial cells also have decreased proliferation in vitro along with the same abnormal proteoglycan and protein expression as seen in patient bladder biopsies in vivo. In addition, BPS/IC cell explants secrete a Frizzled 8-related anti-proliferative factor (APF) (sialyl-Galb1-3GalNAcaO-Thr-Val-Pro-Ala-Ala-Val-Val-Val-Ala) that is not secreted by control cells, is found uniquely in urine from approximately 95% of BPS/IC patients (but not in control urine), and which induces the same abnormalities in proliferation and protein expression in normal bladder epithelial cells as seen in IC cells. The bladder pain plus increased urinary frequency and urgency experienced by BPS/IC patients may therefore result from aberrant bladder epithelial cell gene expression/differentiation that is unique to this syndrome and which results in bladder epithelial thinning or ulceration, leakiness, and abnormal cell protein expression. Because previous microarray experiments showed abnormal BPS/IC bladder epithelial cell expression of several differentiation proteins plus a chromatin remodeling protein, recent studies were performed which determined that BPS/IC cells indeed display an abnormal complement of certain gene regulatory proteins (including specific histone deacetylases (HDACs), transcription factors, and microRNAs) that could explain their abnormal gene expression and differentiation. Therefore, the proposed research will address the hypothesis that the gene expression abnormalities that we and others have described in BPS/IC bladder epithelial cells both in vivo and/or in vitro result from aberrant levels of specific gene regulatory factors. Our preliminary findings of abnormal HDACs, specific transcription factors, and microRNAs in explanted BPS/IC cells will be confirmed using explants from additional BPS/IC patients and controls, and the potential role for these and other regula- tory factors in abnormal specific gene expression determined using Western blot, chromatin immunoprecipita- tion (ChIP), microRNA microarray, and quantitative real-time polymerase chain reaction (qRT-PCR) techniques. The role of each regulatory factor in causing the BPS/IC cell phenotype will be confirmed via loss or gain of function experiments (including siRNA knockdown and transfection). Because APF can induce the same changes in proliferation and permeability, as well as many of the consistent gene expression abnormal- ities found in BPS/IC bladder cells in vivo and/or in vitro, the role of this toxin in regulation of these chromatin- related factors willalso be studied, and the ability of four APF inhibitors (shown previously to normalize proliferation, paracellular permeability, and tight junction protein expression in BPS/IC cells) to normalize chromatin regulatory factors in BPS/IC cells determined. Finally, the ability of a readily availabl, relatively nontoxic HDAC stimulator (theophylline) to normalize gene expression in BPS/IC cells will also be established. These studies should yield valuable information about the mechanism of aberrant gene expression/ differentiation in BPS/IC bladder cells, potentially leading to development of more effective therapies.

描述（由申请人提供）： 膀胱疼痛综合征/间质性膀胱炎（BPS/IC）是一种慢性疼痛膀胱综合征，影响了美国约一百万人的生活。对于退伍军人人口而言，BPS/IC也变得越来越重要，因为据报道，2006年是第四种最常见的泌尿科疾病，并且是所有VA医疗保健使用者中有1.4％的主要诊断（比1999 - 2002年的38％增加了13.7％，原始数量增加了38％，最新数据可用）。 BPS/IC的病因尚不清楚，目前尚无可靠的有效治疗方法。因此，研究这种使人衰弱的疾病的发病机理仍然很重要，以系统地设计有效的，特定的疾病特异性疗法。上皮异常是来自BPS/IC患者的膀胱活检中最一致的发现，包括上皮的剥落，泪水或稀疏，再加上异常的蛋白聚糖和蛋白质表达（包括紧密连接和蛋白质的蛋白质和蛋白质蛋白质和蛋白质蛋白质，Vimentin和uroplakakin iii III）。在体内，外植的BPS/IC膀胱上皮细胞也与体内患者膀胱活检相同的异常蛋白聚糖和蛋白质表达降低。此外，BPS/IC细胞外植物分泌了一个与毛躁相关的抗增殖因子（APF）（siAlyl-galb1-3galnacao-thr-th-th-th-val-pro-ala-ala-ala-ala-ala-val-val-val-ala-ala）分泌，对照细胞分泌，在对照细胞中分泌，在对照细胞中分泌，在大约95％的尿液中发现了尿液和尿液中的尿液和尿液中的尿液，但尿液中的尿液和尿液中的尿液不足。正常膀胱上皮细胞的增殖和蛋白质表达异常，如IC细胞所示。因此，BPS/IC患者经历的膀胱疼痛加上增加的尿频和紧迫性可能是由于该综合征独有的异常上皮细胞基因表达/分化，这会导致膀胱上皮稀薄或溃疡，泄漏，泄漏性，异常细胞蛋白表达。 Because previous microarray experiments showed abnormal BPS/IC bladder epithelial cell expression of several differentiation proteins plus a chromatin remodeling protein, recent studies were performed which determined that BPS/IC cells indeed display an abnormal complement of certain gene regulatory proteins (including specific histone deacetylases (HDACs), transcription factors, and microRNAs) that could explain their abnormal gene expression and分化。因此，拟议的研究将解决以下假设：我们和其他人在体内和/或体外都在BPS/IC膀胱上皮细胞中描述的基因表达异常是由于特定基因调节因素的异常和/或体外所致。我们将使用来自其他BPS/IC患者和对照组的外植体确认我们对植入BPS/IC细胞中异常HDAC，特定的转录因子和microRNA的初步发现，以及这些和其他调节因素的潜在作用，使用蛋白质印刷，量子量和量子量，pronation和Micirorna，micicorna，micirore，micicorna，micirorna，micirore，micirore，micirore，micirorna，micirore，micirore，micirorna，micirorna，micirorna m micimorna m miciper，链反应（QRT-PCR）技术。每个调节因子在引起BPS/IC细胞表型中的作用将通过功能实验的损失或增益（包括siRNA敲低和转染）确认。因为APF可以在体内和/或体外在BPS/IC膀胱细胞中发现的许多一致的基因表达异常诱导相同的变化，以及许多一致的基因表达异常和/或体外，这种毒素在调节这些相关因素的调节中的作用，并在这些相关因素中均具有porter rya的延伸能力，以及以前的plu​​difer parrabife and parceR parcel parceR parceRAIMES，以及正常的正常抑制剂，正常地正常。 BPS/IC细胞中的蛋白质表达）以确定BPS/IC细胞中的染色质调节因子。最后，还将建立易于使用的，相对无毒的HDAC刺激剂（茶碱）在BPS/IC细胞中标准化基因表达的能力。这些研究应产生有关BPS/ IC膀胱细胞中异常基因表达/分化机制的宝贵信息，并有可能导致更有效的疗法的发展。