miR-19a DNA去甲基化及其靶基因GRK6在肠易激综合征内脏痛的作用机制

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by chronic visceral pain in association with altered bowel habits. However, its precise mechanisms of visceral hypersensivity remain elusive, leading to limited and disappointed treatment options. According to the evidence by clinical epidemiologic studies that early life risk factors contribute to IBS in adults, we proposed that epigenetic modification of key nociceptive gene in developmental reprogramming is molecular basis of IBS visceral pain. We have developed and validated neonatal colonic inflammation (NCI) model, a rat model of adult visceral hyperalgesia that mimics some features of human IBS. Through microRNA array analysis, we have screened out a vulnerable development-related microRNA , miR-19a, which was confirmed to be significantly upregualted in dorsal root ganglion (DRG) of NCI rats. Our recent studies showed that miR-19a antagomir could significantly reverse visceral hypersensitivity of NCI rats; several binding sites for translation factor SP1 in CpG island of miR-19a gene promoter were indicated by Gene regulation software; the mRNA level of GRK6, a potential target gene of miR-19a predicted by online software, was significantly downregulated; and GRK6 over-expression lentiviral could significantly reverse the increased excitability of colonic-specific primary sensory neurons from NCI rats. Based on these results, we proposed the following hypothesis: NCI leads to a significant demethylation of CpG island in miR-19a gene promoter and enhanced ability of SP1 to bind the promoter of miR-19a gene, resulting in upregulation of the miR-19a in colonic-specific DRG, then negatively regulates the translation of GRK6, eventually produces chronic visceral hyperalgesia. To test this hypothesis, we propose the following three specific aims: Specific Aim 1: To identify the epigenetic mechanism of miR-19a upregulation in DRG of NCI rats; Specific Aim 2: To explore the contribution of transcript factor SP1 in regulating miR-19a expression; Specific Aim 3: To determine the role of GRK6 regulated by miR-19a in visceral hypersensitivity induce by NCI. We believe that our studies may explain the theory of developmental reprogramming by epigenetic mechanism in chronic pain of functional gastrointestinal disorders. The added knowledge would provide new strategies for treatment of chronic visceral pain in IBS patients.

慢性内脏痛是肠易激综合征(IBS)的临床重要症状之一，发病机制尚未阐明，临床上缺乏有效的治疗手段。已有研究表明发育期不良刺激是IBS内脏痛敏的重要诱因，我们的前期工作发现新生期结肠炎症刺激诱导成年大鼠内脏痛敏，在脊髓背根神经节中发育期易感性基因miR-19a表达上调，痛相关基因G蛋白偶联受体激酶6(GRK6)表达下调。生物信息学预测发现miR-19a启动子区有转录因子SP1的多个结合位点，GRK6是miR-19a可能的靶基因。由此提出假说：发育期炎症刺激引起miR-19a基因启动子区去甲基化，转录因子SP1与miR-19a基因启动子区的结合增强促进miR-19a上调，抑制靶分子GRK6活性，介导内脏痛敏。本课题旨在阐明SP1/miR-19a/GRK6网络调控在慢性内脏痛中的作用机制，为临床治疗提供理论依据和新的治疗靶点。

肠易激综合征（IBS）慢性内脏痛的发病机制不明确，缺乏有效的临床治疗手段，大量研究表明个体发育关键期的不良环境刺激是个体成年后IBS患病的重要启动因素。本研究通过建立新生期结肠炎症刺激组和成熟期结肠炎症刺激组动物模型，通过microRNA芯片检测，在脊髓背根神经节筛选出发育期易感性痛相关基因miR-19a，进一步通过分子生物学及行为学研究表明新生期结肠炎性刺激诱发发育期易感基因miR-19上调，靶向调控G蛋白偶联受体激酶6（GRK6）下调，参与NCI诱导的慢性内脏痛觉过敏。研究结果发现TDG表达上调，引起miR-19a基因启动子区CpG岛去甲基化，可能是其表达上调的重要机制。这些结果初步阐明了microRNA的DNA甲基化机制在慢性内脏痛形成中的作用，为功能性胃肠疾病的“发育源疾病学说”提供理论依据，有望为IBS慢性内脏痛的临床治疗提供新的药物靶点。