Long Noncoding RNA H19 Mediating Alternative Splicing in ALD Pathogenesis

项目摘要 过量饮酒是酒精相关性肝病(ALD)的主要原因。ALD是一种主要的 由于其发病率和流行率的上升，美国的公共卫生问题。更好地理解 ALD的发病机制至关重要，可能为确定潜在的治疗靶点铺平道路。H19是 一种长的非编码RNA；它在酒精喂养的ALD患者的肝脏中高度上调表达 老鼠。然而，H19在ALD发病机制中的作用机制尚未完全阐明。我们最重要的是 本次资助申请的目的是为了了解H19介导的酒精性肝损伤的机制。 针对具体目标1，我们计划确定酒精诱导肝脏H19表达的分子机制 通过DNA甲基化的表观遗传调控和转录调控这两条主要途径 通过小分子异源二聚体(SHP)和早期生长反应1(Egr1)表达。我们还将利用我们独特的 小鼠模型、H19母体特异性基因敲除(H19Mat+/-)和肝脏特异性H19 DNA甲基化结构域 (DMD)基因敲除(H19DMDHep-/-)小鼠，探讨H19功能丧失对肝脏表型的影响。在……里面 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。 我们用RNA免疫沉淀实验筛选了H19相互作用的蛋白，发现了多嘧啶核苷束 结合蛋白1(PTBP1)与H19RNA的N末端结合。PTBP1是一种RNA结合蛋白，主要作用于 作为前体信使核糖核酸(前信使核糖核酸)选择性剪接的抑制调节因子。我们还发现了酒精和H19 PTBP1表达水平降低，选择性剪接事件增加。因此，我们将确定 PTBP1缺乏对乙醇喂养小鼠肝脏表型的影响 特异性基因敲除(Ptbp1Hep-/-)小鼠。此外，我们还发现H19-PTBP1轴参与了细胞的剪接。 其新的靶基因甜菜碱和同型半胱氨酸甲基转移酶(BHMT)，这是一个关键的酶。 蛋氨酸代谢途径。剪接过程导致BHMT蛋白编码变体减少 而BHMT蛋白表达的降低导致蛋氨酸代谢失调，从而 导致酒精性肝损伤。我们将进行几项机械研究，以确定 H19-PTBP1轴在介导BHMT交替剪接中的作用加在一起，我们开发了动物和 细胞模型对H19介导的ALD上下游通路的力学研究 发病机制。这一建议具有重要意义，它可能导致潜在的治疗干预 ALD患者靶向H19-PTBP1-BHMT通路的研究

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.