Long Noncoding RNA H19 Mediating Alternative Splicing in ALD Pathogenesis

长非编码 RNA H19 介导 ALD 发病机制中的选择性剪接

• 批准号: 10717440
• 负责人: Zhihong Yang
• 金额: $ 41.44万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10717440
• 关键词: Alcoholic Liver Diseases; Alcohols; Alleles; Alternative Splicing; Animal Model; Applications Grants; Betaine; Binding; Biological Assay; Birth; Cell model; Chromosome 11; Chromosome 7; Code; DNA; DNA Methylation; Data; Docking; Embryo; Enhancers; Enzymes; Epigenetic Process; Ethanol; Event; Gene Cluster; Genes; Goals; Growth; H19 RNA; H19 gene; Heavy Drinking; Hepatic; Hepatocyte; Homocysteine S-methyltransferase; Human; Immunoprecipitation; Incidence; Inherited; Knock-out; Knockout Mice; Liver; Liver diseases; Mediating; Messenger RNA; Methionine Metabolism Pathway; Methylation; Molecular; Mus; N-terminal; Pathogenesis; Pathway interactions; Patients; Phenotype; Polypyrimidine Tract-Binding Protein; Prevalence; Process; Protein Methyltransferases; Proteins; Public Health; RNA; RNA Splicing; RNA-Binding Proteins; Regulation; Role; Sampling; Structure; Testing; Therapeutic Intervention; Tissues; Transcriptional Regulation; United States; Untranslated RNA; Variant; differential expression; epigenetic regulation; experimental study; gene conservation; imprint; mRNA Expression; mRNA Precursor; mouse model; novel; overexpression; paternal imprint; promoter; protein expression; response; therapeutic target

Project Summary Excessive alcohol consumption is a leading cause of alcohol-associated liver disease (ALD). ALD is a major public health issue in the US due to its rising incidence and prevalence. A better understanding of the mechanism of ALD pathogenesis is critical and may pave a way to identify potential therapeutic targets. H19 is a long noncoding RNA; its expression is highly upregulated in the liver of patients with ALD and ethanol-fed mice. However, the mechanism of H19 in ALD pathogenesis has not been fully explored. Our overarching objective of this grant application is to understand the mechanism of H19 mediated alcohol-induced liver injury. For specific aim#1, we plan to determine the molecular mechanism on alcohol induced hepatic H19 expression by focusing on two major pathways, epigenetic regulation via DNA methylation and transcriptional regulation by small heterodimer partner (SHP) and Early Growth Response 1 (EGR1). We will also employ our unique mouse models, H19 maternal specific knockout (H19Mat+/-) and liver specific H19 DNA methylation domain (DMD) knockout (H19DMDHep-/-) mice, to explore how loss of H19 function effects the hepatic phenotypes. In specific aim#2, we will determine the molecular mechanism of how H19 mediates alcohol-induced liver injury. We screened the H19 interacted proteins using RNA immunoprecipitation assay and found polypyrimidine tract binding protein 1 (PTBP1) binds to N-terminal of H19 RNA. PTBP1 is an RNA-binding protein to act primarily as repressive regulator of precursor mRNA (pre-mRNA) alternative splicing. We also found alcohol and H19 reduced PTBP1 expression levels and increased alternative splicing events. Therefore, we will determine the effect of PTBP1 deficiency on hepatic phenotypes in ethanol-fed mice using our newly generated PTBP1 liver specific knock out (Ptbp1Hep-/-) mice. Additionally, we identified that H19-PTBP1 axis mediates the splicing of its novel target gene betaine and homocysteine methyltransferase (BHMT), which was a critical enzyme in the methionine metabolism pathway. The splicing process led to a decrease in the BHMT protein coding variant and the reduction in BHMT protein expression led to a dysregulation of methionine metabolism, which contributed to alcohol induced liver injury. We will perform several mechanistic studies to determine the role of H19-PTBP1 axis in mediating BHMT alternate splicing. Taken together, we have developed animal and cellular models to mechanistically study both up and downstream pathways of H19-mediated ALD pathogenesis. This proposal is of significance and it may lead to potential therapeutic interventions by targeting H19-PTBP1-BHMT pathway in patients with ALD.

项目概要 过量饮酒是酒精相关性肝病（ALD）的主要原因。 ALD 是一个主要 由于其发病率和患病率不断上升，已成为美国的公共卫生问题。更好地理解 ALD 发病机制至关重要，可能为识别潜在治疗靶点铺平道路。 H19 是 长非编码RNA；它的表达在 ALD 和乙醇喂养患者的肝脏中高度上调 老鼠。然而，H19在ALD发病机制中的机制尚未得到充分探索。我们的首要任务 本次资助申请的目的是了解 H19 介导的酒精性肝损伤的机制。 对于具体目标#1，我们计划确定酒精诱导肝脏 H19 表达的分子机制 重点关注两条主要途径：通过 DNA 甲基化进行的表观遗传调控和转录调控 由小异二聚体伴侣 (SHP) 和早期生长反应 1 (EGR1) 产生。我们还将运用我们独特的 小鼠模型、H19 母体特异性敲除 (H19Mat+/-) 和肝脏特异性 H19 DNA 甲基化结构域 (DMD) 敲除 (H19DMDHep-/-) 小鼠，探索 H19 功能丧失如何影响肝脏表型。在 具体目标#2，我们将确定H19如何介导酒精性肝损伤的分子机制。 我们利用RNA免疫沉淀法筛选了H19相互作用蛋白，发现多聚嘧啶束 结合蛋白 1 (PTBP1) 与 H19 RNA 的 N 末端结合。 PTBP1 是一种 RNA 结合蛋白，主要作用 作为前体 mRNA (pre-mRNA) 选择性剪接的抑制性调节因子。我们还发现了酒精和 H19 降低 PTBP1 表达水平并增加选择性剪接事件。因此，我们将确定 使用我们新生成的 PTBP1 肝脏，研究 PTBP1 缺乏对乙醇喂养小鼠肝脏表型的影响 特异性敲除 (Ptbp1Hep-/-) 小鼠。此外，我们还发现 H19-PTBP1 轴介导 其新靶基因甜菜碱和同型半胱氨酸甲基转移酶（BHMT）是 蛋氨酸代谢途径。剪接过程导致BHMT蛋白编码变体减少 BHMT 蛋白表达的减少导致蛋氨酸代谢失调， 导致酒精性肝损伤。我们将进行几项机制研究以确定 H19-PTBP1 轴介导 BHMT 交替剪接。总而言之，我们开发了动物和 用于机械研究 H19 介导的 ALD 的上下游途径的细胞模型 发病。该提议具有重要意义，可能会导致潜在的治疗干预措施 靶向 ALD 患者的 H19-PTBP1-BHMT 通路。