SRSF3 Loss and Hepatocellular Carcinoma

SRSF3 缺失与肝细胞癌

基本信息

批准号：
9205453
负责人：
NICHOLAS J WEBSTER
金额：
--
依托单位：
VA SAN DIEGO HEALTHCARE SYSTEM
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-01-01 至 2019-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9205453
关键词：
Address Age Aging Alternative Splicing American Cancer Society Apoptosis Area Binding Biological Biological Process Cancer Center Carcinoma Cause of Death Cell Cycle Cell Proliferation Cells Chronic Cirrhosis Clinical Code DNA Damage DNA Methylation Data Defect Dependence Dependency Diagnosis Disease Epigenetic Process Event Excision Exons Experimental Designs Family member Fibronectin Receptors Fibronectins Fibrosis Functional disorder General Population Genes Genetic Genetic Transcription Hepatitis B Hepatitis C Hepatocarcinogenesis Hepatocyte Human IGF2 gene INSR gene Immunohistochemistry In Vitro Incidence Individual Insulin Receptor Investigation Knock-out Knockout Mice Knowledge Korea Lead Lesion Liver Liver diseases Lower Organism Maintenance Malignant Neoplasms Malignant neoplasm of liver Messenger RNA Metastatic to Methylation Modeling Molecular Mus Mutation Normal tissue morphology Oncogenic Oxidative Stress Pathogenesis Pathology Pathway interactions Patients Pharmacology Phenotype Physiological Poison Population Predisposition Primary carcinoma of the liver cells Process Proteins RNA Primary Transcript RNA Splicing Receptor Gene Risk Factors Role Sampling Series Site Somatic Mutation Spliced Genes Steatohepatitis Stress Testing Transfection Tumor Suppressor Genes Validation Variant Veterans Vietnam autocrine base biobank carcinogenesis crosslinking and immunoprecipitation sequencing exon skipping experimental study hepatoma cell in vivo insight liver injury mouse model neoplastic neoplastic cell non-alcoholic fatty liver nonalcoholic steatohepatitis novel therapeutic intervention overexpression patient population prevent promoter public health relevance therapeutic development tool transcriptome transcriptome sequencing tumor tumor growth

项目摘要

DESCRIPTION (provided by applicant): RNA splicing is the process of removal of intronic sequences from the primary RNA transcript before the final mRNA is generated. Unlike lower organisms, the vast majority of mammalian genes are spliced. Most genes give rise to multiple mRNAs resulting from differential promoters or termination sequences, or the use of alternative exons. In the broader clinical picture, alternative splicing is an important new area of investigation. Alterations in RNA splicing have now been found in many cancers. As cancer is a leading cause of death among the VA population, understanding how these oncogenic splice variants occur and how they contribute to carcinogenesis is paramount to our understanding of the pathogenesis of the disease and our discovery of novel therapeutic approaches. We have recent data in mice that eliminating a particular RNA splicing factor causes chronic liver damage due to hepatocyte apoptosis and compensatory proliferation. As the mice age, their livers show steatohepatitis and fibrosis, and eventually 100% of the mice succumb to hepatocellular carcinoma (HCC). The loss of the splicing factor does not cause tumors but rather creates a pre-disposition to cancer, similar to a tumor suppressor gene. We believe this model has many parallels to human HCC and can provide great insights into the pathogenesis of this disease that could guide therapeutic development. Based on extensive preliminary data on our splicing factor knockout mouse, we are proposing a comprehensive series of experiments to understand how loss of a single splicing factor in the liver can lead to metastatic hepatocellular carcinoma. These studies will address key questions concerning the fundamental biological process of carcinogenesis and will integrate cell and molecular biological experiments with physiological studies in mice lacking specific splicing factors in liver. We will establish the relevance of this model for human liver cancer by assessing how frequently SRSF3 loss occurs in human HCC, whether loss occurs in all HCC or particular subtypes, and whether loss occurs in early-stage liver disease such as NAFLD, NASH or cirrhosis. In the mouse, loss of SRSF3 predisposes to HCC but not every cell becomes transformed, suggesting that somatic mutations or epigenetic changes must be acquired to produce the carcinoma phenotype. We will attempt to identify these secondary genetic changes by sequencing somatic mutations in the mouse tumors, or assessing changes in CpG methylation in differentially-methylated regions. We will also test whether these genetic changes allow the tumors to become independent of SRSF3 status by restoring SRSF3 expression in the tumor cells. We investigate the molecular mechanisms underlying the aberrant RNA splicing and the associated cellular changes using a combination of CLIP-seq and transcriptome sequencing and splice junction arrays to identify the direct targets for SRSF3 in the hepatocyte. These targets will be tested for SRSF3-dependence using in vitro cultured human hepatoma cells. We will also test a number of SRSF3 mutations have been identified in a variety of cancers including HCC. We will test two potential mechanisms that may contribute to apoptosis, fibrosis, and cell proliferation. We are attempting to connect changes in splicing of the fibronectin and insulin receptor genes with specific intermediate phenotypes, and with tumor growth. These two genes are direct targets for SRSF3 and their splicing is altered in the knockout mouse. We will test whether miss-splicing of the fibronectin gene causes oxidative stress and fibrosis, and whether miss-splicing of the insulin receptor gene allows IGF2 to stimulate tumor cell proliferation and prevent the normal terminal endo-replication of the hepatocyte. These experiments will combine new genetic knockouts with pharmacological stimulation or inhibition of individual pathways. Ultimately, we will test whether these two pathways contribute to hepatocellular carcinogenesis.

描述（由申请人提供）： RNA剪接是在产生最终mRNA之前从初级RNA转录物中去除内含子序列的过程。与较低的生物体不同，绝大多数哺乳动物基因是剪接的。大多数基因产生由差分启动子或终止序列或替代外显子产生的多个mRNA。在更广泛的临床情况下，替代剪接是一个重要的新调查领域。现在在许多癌症中发现了RNA剪接的改变。由于癌症是VA人群中死亡的主要原因，因此了解这些致癌剪接变体是如何发生的，以及它们如何对癌变做出贡献，对我们对疾病发病机理的理解以及我们发现新型治疗方法的理解至关重要。我们在小鼠中有最近的数据，这些数据消除了特定的RNA剪接因子会导致肝细胞凋亡和补偿性增殖引起的慢性肝损伤。随着小鼠的年龄，他们的生活显示出脂肪性肝炎和纤维化，最终100％的小鼠屈服于肝细胞癌（HCC）。剪接因子的丧失不会引起肿瘤，而是引起癌症的前置态，类似于肿瘤抑制基因。我们认为，该模型与人类HCC有许多相似之处，可以为该疾病的发病机理提供很好的见解，可以指导热发育。基于有关我们的剪接因子基因敲除小鼠的广泛初步数据，我们提出了一系列全面的实验，以了解肝脏中单个剪接因子的损失如何导致转移性肝细胞癌。这些研究将解决有关癌变基本生物学过程的关键问题，并将细胞和分子生物学实验与缺乏肝脏中缺乏特定剪接因子的小鼠的物理研究整合。我们将通过评估人类HCC中的SRSF3损失的频率，是否发生在所有HCC或特定亚型中，以及是否发生在NAFLD，NAFLD，NASH或CIRRHOSOS等早期肝病中。在小鼠中，SRSF3损失易于HCC，但并非每个细胞都会转化，这表明必须获得体细胞突变或表观遗传变化以产生癌表型。我们将尝试通过对小鼠肿瘤中的体细胞突变进行测序，或评估不同甲基化区域中CpG甲基化的变化来鉴定这些二次遗传变化。我们还将测试这些遗传变化是否使肿瘤通过恢复肿瘤细胞中的SRSF3表达而独立于SRSF3状态。我们使用夹子序列和转录组测序和剪接连接阵列的组合研究了异常RNA剪接和相关细胞变化的分子机制，以识别肝细胞中SRSF3的直接靶标。这些靶标将使用体外培养的人肝癌细胞测试SRSF3依赖性。我们还将在包括HCC在内的各种癌症中鉴定出许多SRSF3突变。我们将测试可能导致凋亡，纤维化和细胞增殖的两种潜在机制。我们正在尝试连接拼接的变化纤连蛋白和胰岛素受体基因具有特定的中间表型，并具有肿瘤生长。这两个基因是SRSF3的直接靶标，其剪接在敲除小鼠中发生了变化。我们将测试纤连蛋白基因的遗漏是否会导致氧化应激和纤维化，以及胰岛素受体基因的遗漏是否允许IGF2允许IGF2刺激肿瘤细胞的增殖并防止肝细胞的正常末端内部再生。实验将将新的遗传敲除与药物刺激或抑制单个途径相结合。最终，我们将测试这两种途径是否有助于肝细胞癌。