LncRNA H19 in Cholestatic Liver Diseases

原发性硬化性胆管炎(PSC)是主要的胆汁淤积性肝病，发病率和死亡率均较高。 由胆汁形成或流动障碍引起的胆管炎症和纤维化损伤是主要的 PSC的特性。哺乳动物基因组的大部分是非蛋白质编码序列。少于 人类基因组的2%由蛋白质编码基因组成。转录的哺乳动物中的很大一部分 基因组是非编码RNA(NcRNAs)，包括长非编码RNA(LncRNAs)、环状RNA(CircRNAs)、 和microRNA(MiRNAs)，但只有少数几个被结构注释。它的生物学功能 NcRNA在很大程度上仍不为人所知。高通量RNA测序(RNAseq)和CircRNA- 特定的生物信息学算法已经识别出数千个具有组织特异性表达模式的CircRNA。 然而，CircRNAs在疾病中，特别是在肝病中的相关性和功能仍有待研究。 下定决心。我们以前的研究报告了结合初级胆汁酸(CBA)，牛磺胆酸(TCA)， 激活鞘氨醇-1磷酸受体2(S1PR2)，诱导胆管细胞H19表达。TCA- H19的诱导上调是胆汁淤积性肝损伤的主要促进剂 巨噬细胞和肝星状细胞(HSCs)。我们使用Arraystar CircRNAseq的新初步数据确认 33个重叠的CircRNA，在mdr2-/-小鼠中显著上调，在mdr2-/-/H19- /-老鼠。其中，MMU_CircRNA_26644(CircRNA-Edil3)，由表皮生长因子样蛋白编码 重复和盘状I样结构域3(Edil3)基因，是mdr2-/-中下调最显著的CircRNA /H19-/-小鼠。序列分析表明，CircRNA-Edil3可能是miRNA-215-3p的“海绵”， MiRNA-129-5p和miRNA-3075-3p。TargetScan分析表明高迁移率族盒蛋白1 (HMGB1)、AF4/FMR2家族成员3(Aff3)和Edil3是miRNA-215-3p、miRNA-129-5p的潜在靶点 和miRNA-3075。根据我们公布的结果和令人振奋的新的初步数据，我们假设 H19诱导的CircRNA-Edil3上调在隔离性胆汁淤积性肝损伤中起关键作用 MiRNAs。为了检验我们的假设，本文提出了两个具体目标。目的1.研究H19诱导的细胞毒作用 CircRNA-Edil3对胆管细胞增殖、衰老和肝脏炎症的调节作用 胆汁淤积性肝损伤小鼠模型和人PSC患者的肝脏和血清样本。目标2：实现 确定H19诱导的CircRNA-Edil3促进胆汁淤积性肝损伤的机制。我们会 使用多种新建立的方法和动物模型来检验我们的假设。完成这些工作 特异性靶点应阐明lncRNAH19介导的潜在细胞/分子机制 研究胆汁淤积性肝病的进展并确定新的诊断和治疗靶点。因为这些影响 胆汁淤积是深刻而广泛的，导致肝脏疾病和全身疾病的恶化， 这项建议的主题是及时的，重要的，并具有直接的临床应用。

Primary sclerosing cholangitis (PSC) is the major cholestatic liver disease with high morbidity and mortality. Inflammation and fibrotic injury of the bile ducts due to impairment of bile formation or flow represent the major characteristics of PSC. The majority of the mammalian genome are non-protein-coding sequences. Less than 2% of the human genome is made up of protein-coding genes. A large fraction of the transcribed mammalian genome is noncoding RNAs (ncRNAs), including long noncoding RNAs (lncRNAs), circular RNA (circRNAs), and microRNA (miRNAs), but only a few of them have been structurally annotated. The biological functions of ncRNAs remain largely unknown. Recent advances in high-throughput RNA sequencing (RNAseq) and circRNA- specific bioinformatics algorithms have identified thousands of circRNAs with tissue-specific expression patterns. However, the relevance and function of circRNAs in disease, especially in liver diseases, remain to be determined. Our previous studies reported that the conjugated primary bile acid (CBA), taurocholic acid (TCA), activated sphingosine-1phosphate receptor 2 (S1PR2) and induced H19 expression in cholangiocytes. TCA- induced upregulation of H19 is a major promoter of cholestatic liver injury by activating inflammatory macrophages and hepatic stellate cells (HSCs). Our new preliminary data using Arraystar circRNAseq identified 33 overlapping circRNAs, which were significantly upregulated in Mdr2-/- mice and downregulated in Mdr2-/-/H19- /- mice. Among them, mmu_circRNA_26644 (circRNA-Edil3), which is encoded by epidermal growth factor-like repeats and discoidin I-like domains 3 (Edil3) gene, is the most significantly downregulated circRNA in Mdr2-/- /H19-/- mice. Sequence analysis suggested that circRNA-Edil3 may serve as a "sponge" for miRNA-215-3p, miRNA-129-5p and miRNA-3075-3p. TargetScan analysis suggested that High Mobility Group Box Protein 1 (HMGB1), AF4/FMR2 Family Member 3 (Aff3) and Edil3 are potential targets of miRNA-215-3p, miRNA-129-5p and miRNA-3075. Based on our published results and exciting new preliminary data, we HYPOTHESIZE that H19-induced upregulation of circRNA-Edil3 plays a critical role in cholestatic liver injury by sequestering miRNAs. Two specific aims are proposed to test our hypothesis. Aim 1. To examine the role of H19-induced circRNA-Edil3 in regulating cholangiocyte proliferation, senescence and hepatic inflammation using cholestatic liver injury mouse models and human PSC patient liver and serum samples. Aim 2: To identify the mechanisms by which H19-induced circRNA-Edil3 promotes cholestatic liver injury. We will use multiple newly-established approaches and animal models to test our hypothesis. Completion of these specific aims should elucidate the potential cellular/molecular mechanisms involved in lncRNA H19-mediated progression of cholestatic liver diseases and identify novel diagnostic and therapeutic targets. Since the effects of cholestasis are profound and widespread, leading to worsening liver diseases and systemic illness, the subject matter of this proposal is timely, important, and has direct clinical application.