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发病机制的研究是至关重要的，并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;其表达在ALD和乙醇喂养患者的肝脏中高度上调 小鼠然而，H19在ALD发病机制中的作用机制尚未完全阐明。我们的总体 本基金申请的目的是了解H19介导的酒精性肝损伤的机制。 对于特定的目标#1，我们计划确定酒精诱导的肝H19表达的分子机制 通过关注两个主要途径，即通过DNA甲基化的表观遗传调控和转录调控， 小异源二聚体伴侣（SHP）和早期生长反应1（EGR 1）。我们还将利用我们独特的 小鼠模型，H19母体特异性敲除（H19 Mat +/-）和肝脏特异性H19 DNA甲基化结构域 (DMD)敲除（H19 DMDHep-/-）小鼠，以探索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如何介导酒精诱导的肝损伤的分子机制。 我们用RNA免疫沉淀法筛选了H19相互作用蛋白，发现了多聚嘧啶序列 结合蛋白1（PTBP 1）与H19 RNA的N末端结合。PTBP 1是一种RNA结合蛋白， 作为前体mRNA（pre-mRNA）选择性剪接的抑制性调节剂。我们还发现了酒精和H19 PTBP 1表达水平降低和可变剪接事件增加。因此，我们将确定 PTBP 1缺陷对使用我们新生成的PTBP 1肝脏的乙醇喂养小鼠的肝脏表型的影响 特异性敲除（Ptbp 1Hep-/-）小鼠。此外，我们发现H19-PTBP 1轴介导了 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 BHMT蛋白表达的减少导致甲硫氨酸代谢的失调， 导致酒精引起的肝损伤。我们将进行几项机制研究，以确定 H19-PTBP 1轴介导BHMT可变剪接。总之，我们已经开发了动物和 细胞模型，以机械地研究H19介导的ALD的上游和下游途径 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， 靶向H19-PTBP 1-BHMT通路的ALD患者。

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.