Mechanisms of Altered Gastrointestinal Dysfunction

描述（申请人提供）：肠易激综合征（IBS）患者患有慢性腹痛和相关的胃肠道症状-腹泻，便秘，腹胀和紧迫感。那些症状严重的人也可能对伤害性刺激有内脏超敏反应，这表明异常的神经疼痛处理机制。事实上，我们发现一个重要的IBS患者子集（约35%）对有害的实验刺激（例如，结肠扩张）;但机制尚不清楚。我们在之前NIH支持的研究（R 01 NS 053090）中发现了该机制的线索;我们在该亚组的结肠组织中鉴定了独特的microRNA（miRNAs）。在这里，我们将通过验证以下假设来追踪这一线索：关键miRNA的异常表达使下游miRNA靶点的表达失调（阿片受体; NMDA受体），这有助于IBS患者的内脏高敏感性。 为了确定独特的miRNA在下游受体靶基因的表观遗传调控中的细胞内和细胞间作用，我们的实验室开发了创新的方法。这些方法将锁核酸（LNA）修饰的寡核苷酸探针的独特miRNA识别特性与使用酪胺信号放大（TSA）技术的荧光原位杂交（FISH）结合联合收割机。我们将使用体内鞘内注射miRNA抑制剂（LNA）或miRNA模拟物（前体）来在我们验证的内脏高敏感性大鼠模型中实验性地调节靶向阿片样物质（Aim 1）和谷氨酸（Aim 2）受体的特异性miRNA的表达（Zhou等人，Pain，2008）。因此，我们将使用多种策略的组合来开发一个全面的高置信度的阿片类和谷氨酰胺受体的miRNA靶向网络模型。 在目的1中，我们将确定增加的miR-29 a/B簇和miR-29 B-1- 5 p表达是否通过下调阿片样物质信号通路增强内脏高敏感性。在目标2中，我们将确定降低的miR-23 a/B簇和125 B-1- 3 p表达是否通过上调谷氨酸信号传导机制促进内脏高敏感性。我们提出的研究应该确定异常表达的miRNA的关键贡献，这些miRNA靶向具有内脏高敏感性的IBS患者的肠组织中的下游阿片样物质和谷氨酸受体。这将弥补我们对导致患有常见功能性GI疾病的患者的慢性疼痛和改变的内脏伤害感受的伤害性通路的失调的潜在机制的知识的重要空白。更重要的是，这些结果应该通过开发合成的寡核苷酸来开发创新的治疗策略，这些寡核苷酸抑制或模拟特定的miRNA，这些miRNA调节下游靶基因（例如阿片样物质或谷氨酸受体）的表观遗传调控，这些下游靶基因驱动内脏伤害感受。建议的转化研究的最终目标是开发新的治疗IBS和其他功能性GI疾病的患者的疗法。

DESCRIPTION (provided by applicant): Patients with irritable bowel syndrome (IBS) suffer from chronic abdominal pain and associated GI symptoms - diarrhea, constipation, bloating & urgency. Those with severe symptoms may also have visceral hypersensitivity to nociceptive stimuli, suggesting aberrant neural pain processing mechanisms. Indeed, we found a significant subset (~35%) of IBS patients with visceral hypersensitivity to noxious experimental stimuli (e.g., colonic distension); but the mechanism was unclear. We found a clue to that mechanism during our previous NIH-supported study (R01NS053090); we identified unique microRNAs (miRNAs) in colon tissues of this subset. Here we will follow up on that clue by testing the following hypothesis: Aberrant expression of key miRNAs dysregulates expression of downstream miRNA targets (opioid receptors; NMDA receptors) which contributes to visceral hypersensitivity in IBS patients. To determine the intra- and inter-cellular roles of unique miRNAs in the epigenetic regulation of down- stream receptor target genes, our lab has developed innovative methods. These methods combine unique miRNA recognition properties of locked nucleic acid (LNA) modified oligonucleotide probes with fluorescence in situ hybridization (FISH) using tyramide signal amplification (TSA) technology. We will use in vivo intrathecal injection of miRNA inhibitors (LNAs) or miRNA mimics (precursors) to experimentally regulate expression of specific miRNAs that target opioid (Aim 1) and glutamate (Aim 2) receptors in our validated rat model of visceral hypersensitivity (Zhou et al., Pain, 2008). Thus, we will use a combination of strategies to develop a comprehensive high-confidence model of miRNA targeting networks for opioid and glutamine receptors. In Aim 1 we will determine whether increased miR-29a/b cluster and miR-29b-1-5p expression enhances visceral hypersensitivity via downregulation of opioid signaling pathways. In Aim 2, we will determine whether decreased miR-23a/b cluster and 125b-1-3p expression facilitates visceral hypersensitivity via upregulation of glutamate signaling mechanisms. Our proposed studies should identify critical contributions of aberrantly expressed miRNAs that target downstream opioid and glutamate receptors in intestinal tissues of IBS patients with visceral hypersensitivity This will bridge an important gap in our knowledge of the underlying mechanisms of dysregulation of nociceptive pathways that lead to chronic pain and altered visceral nociception in patients who suffer from common functional GI disorders. More importantly, the results should lead to the development of innovative therapeutic strategies via development of synthesized oligonucleotides that inhibit or mimic specific miRNAs that modulate epigenetic regulation of down-stream target genes (e.g. opioid or glutamate receptors) that drive visceral nociception. The ultimate goal of the proposed translational studies is to develop new therapies for treating our patients with IBS and other functional GI disorders.