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.

项目摘要 过度饮酒是酒精相关肝病（ALD）的主要原因。 ALD是主要的 由于其发病率和流行率上升，美国的公共卫生问题。更好地理解 ALD发病机理的机理至关重要，可以铺平一种识别潜在治疗靶标的方法。 H19是 长的非编码RNA；它的表达在ALD和乙醇喂养的患者的肝脏中高度上调 老鼠。但是，尚未完全探索H19在ALD发病机理中的机制。我们的总体 该赠款应用的目的是了解H19介导的酒精诱导的肝损伤的机制。 对于特定目标＃1，我们计划确定酒精诱导的肝H19表达的分子机制 通过专注于两种主要途径，通过DNA甲基化和转录调节的表观遗传调节 由小型异二聚体合作伙伴（SHP）和早期生长反应1（EGR1）。我们还将利用我们的独特 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，以探索H19功能的损失如何影响肝表型。在 具体的目标＃2，我们将确定H19如何介导酒精诱导的肝损伤的分子机制。 我们使用RNA免疫沉淀测定法筛选了H19相互作用的蛋白 结合蛋白1（PTBP1）与H19 RNA的N末端结合。 PTBP1是一种主要作用的RNA结合蛋白 作为前体mRNA（前mRNA）替代剪接的抑制性调节剂。我们还发现酒精和H19 降低了PTBP1表达水平并增加了替代剪接事件。因此，我们将确定 PTBP1缺乏对乙醇喂养小鼠肝表型的影响，使用我们新生成的PTBP1肝脏 特定的敲除（PTBP1HEP - / - ）小鼠。此外，我们确定H19-PTBP1轴介导 它的新型靶基因甜菜碱和同型半胱氨酸甲基转移酶（BHMT），这是该酶的一种关键酶 蛋氨酸代谢途径。剪接过程导致BHMT蛋白质编码变体的减少 BHMT蛋白表达的降低导致蛋氨酸代谢失调，这 导致酒精引起的肝损伤。我们将进行几项机械研究，以确定 H19-PTBP1轴介导BHMT替代剪接。综上所述，我们已经开发了动物， 细胞模型，用于机械地研究H19介导的ALD的上和下游途径 发病。该建议具有重要意义，可能会导致潜在的治疗干预措施 针对ALD患者的H19-PTBP1-BHMT途径。