Aberrant splicing of the E-cadherin gene.

E-钙粘蛋白基因的异常剪接。

• 批准号: 8196321
• 负责人: Sanjai Sharma
• 金额: --
• 依托单位: VA GREATER LOS ANGELES HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-10-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8196321
• 关键词: Address; Age; Age-Months; Alternative Splicing; Apoptosis; B-Lymphocytes; Binding; Biological; Biological Assay; Biology; Blood; Cell Nucleus; Cells; Cessation of life; Chronic; Chronic Lymphocytic Leukemia; Codon Nucleotides; Data; Degradation Pathway; Detection; Diagnosis; Disease; E-Cadherin; Ectopic Expression; Elements; Emetine; Exons; Frequencies; Gene Mutation; Gene Silencing; Genes; Genetic Transcription; Goals; Incidence; Indium; Indolent; Investigation; Journals; Lead; Leukemic Cell; Loss of E-cadherin Expression; Malignant - descriptor; Manuscripts; Mediating; Messenger RNA; Molecular; Molecular Abnormality; Mus; Mutation; Nonsense Codon; Oligonucleotides; Outcome; Pathway interactions; Patients; Pattern; Play; Population; Premalignant; Process; Publications; RNA; RNA Splicing; Relative (related person); Reporter; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Role; Running; Sampling; Signal Pathway; Signal Transduction; Specimen; Techniques; Tertiary Protein Structure; Testing; Therapeutic Intervention; Time; Transcript; Transgenic Mice; Translations; Tumor Suppressor Genes; Up-Regulation; Veterans; Western World; Work; abstracting; base; beta catenin; cancer type; cohort; deletion analysis; exon skipping; inhibitor/antagonist; leukemia; leukemogenesis; loss of function; male; migration; mouse model; novel; outcome forecast; overexpression; patient population; public health relevance; research study; tumorigenesis

DESCRIPTION (provided by applicant): Project abstract: Aberrant splicing of the E-cadherin gene. Chronic lymphocytic leukemia (CLL) is the most common leukemia and an incurable disease. To identify novel genetic mutations in chronic lymphocytic leukemia cells, we used a microarray technique to screen for genes, which harbor premature termination codons (PTCs). These PTC mutations contribute to oncogenesis by expressing a truncated non-functional mRNA, which is degraded by the nonsense-mediated degradation (NMD) pathway resulting in loss of expression. Among the genes with highest upregulation, we identified the E-cadherin gene, a tumor suppressor gene. The PTC is in an alternatively spliced of E-cadherin transcript which is stabilized when the NMD pathway is blocked. This transcript of E-cadherin completely lacks exon 11 (exon skipping), resulting in a frameshift and a PTC codon in exon 12. RT-PCR analysis demonstrated that the exon 11 skipping occurs in normal B cells as well but at a much lower frequency. We also found a significant decrease in total wild type E-cadherin RNA expression in 62% of CLL specimens (n=35). Furthermore, there was an inverse correlation (p=0.018) between high aberrant transcript and lower E-cadherin expression in CLL specimens. Our results suggest a novel mechanism of E-cadherin gene loss of function in which the trans- factors/splicing factors in CLL cells induce an increased non-productive/aberrant splicing of this tumor suppressor gene. A downstream effect of loss of E-cadherin is the activation of the wnt pathway as beta-catenin is now able to translocate to the nucleus. This pathway is active in CLL cells and our data using reporter assays confirms this finding. Our preliminary experiments also show the reporter activity is higher in CLL specimens with lower E-cadherin expression and the reporter activity can be reversed with ectopic expression of E-cadherin alone. In specific aim 1, we will study the significance of E-cadherin loss of expression in CLL cells. E-cadherin loss can result in activation wnt- -catenin pathway along which can also be activated by other wnt pathway activators (e.g. wnt and frizzled genes). We will determine the relative role of E-cadherin loss and the wnt/frizzled genes in wnt pathway upregulation. Experiments will be performed to inhibit this pathway by E-cadherin re-expression and its subsequent effects on apoptosis, migration and invasion. In specific aim 2, the cis- and trans- elements involved in the aberrant splicing of exon 11 will be studied. With the help of a minigene construct, the cis-elements involved in aberrant splicing will be analyzed by deletions and an antisense oligo strategy. The trans-factors or the splicing factors involved in aberrant splicing will be identified by studying the differentially expressed splicing factors in CLL and normal B cells. The role of these splicing factors will be confirmed in minigene experiments by expressing or inactivating the factors and determining their effect on aberrant splicing. Our goal here is to identify the relevant cis-elements and the trans-factors which bind them so that attempts can be made to modify the aberrant splicing process and restore E-cadherin expression. PUBLIC HEALTH RELEVANCE: Aberrant splicing of the E-cadherin gene. We are studying the most common leukemia in the western world, chronic lymphocytic leukemia (CLL). The incidence of this leukemia increases with age and its incidence is higher in males. For these reasons, CLL is a common diagnosis in the Veteran patient population. Our ability to predict outcome in CLL patients is still limited and therefore, we are unable to tailor specific treatment to CLL patients. Identification of new genetic abnormalities in CLL will be helpful to determine prognosis and open new avenues for treatment. We have identified genetic abnormalities in a gene called E-cadherin in CLL which is a well known gene in other types of cancers. We have performed extensive experimental work, which supports our contention that E-cadherin is an important gene in CLL. Our proposal will further study the role of this gene in CLL.

描述（由申请人提供）： 项目摘要：E-钙粘蛋白基因的异常剪接。慢性淋巴细胞白血病（CLL）是最常见的白血病，也是一种无法治愈的疾病。为了识别慢性淋巴细胞白血病细胞中的新基因突变，我们使用微阵列技术来筛选含有提前终止密码子（PTC）的基因。这些PTC突变通过表达截短的非功能性mRNA而促进肿瘤发生，所述截短的非功能性mRNA通过无义介导的降解（NMD）途径降解，导致表达丧失。在上调最高的基因中，我们鉴定了E-钙粘蛋白基因，一种肿瘤抑制基因。PTC是E-钙粘蛋白转录物的选择性剪接，当NMD途径被阻断时，该转录物被稳定。E-cadherin的转录本完全缺乏外显子11（外显子跳跃），导致外显子12中的移码和PTC密码子。RT-PCR分析表明，外显子11跳跃也发生在正常B细胞中，但频率要低得多。我们还发现在62%的CLL标本中总野生型E-钙粘蛋白RNA表达显著降低（n = 35）。此外，CLL标本中高异常转录本和低E-钙粘蛋白表达之间存在负相关（p = 0.018）。我们的研究结果表明，E-钙粘蛋白基因功能丧失的一种新机制，其中CLL细胞中的反式因子/剪接因子诱导该肿瘤抑制基因的非生产性/异常剪接增加。E-钙粘蛋白缺失的下游效应是wnt途径的激活，因为β-连环蛋白现在能够易位到细胞核。该途径在CLL细胞中是活跃的，我们使用报告基因测定的数据证实了这一发现。我们的初步实验还表明，在CLL标本与较低的E-钙粘蛋白表达的报告活性较高，报告活性可以逆转与异位表达的E-钙粘蛋白单独。在具体目标1中，我们将研究E-钙粘蛋白在CLL细胞中表达缺失的意义。E-钙粘蛋白缺失可导致wnt-β-连环蛋白途径沿着活化，其也可被其它wnt途径活化剂（例如wnt和frizzled基因）活化。我们将确定E-cadherin丢失和wnt/frizzled基因在wnt通路上调中的相对作用。将进行实验以通过E-cadherin再表达及其对细胞凋亡、迁移和侵袭的后续影响来抑制该途径。在具体目标2中，将研究涉及外显子11的异常剪接的顺式和反式元件。在小基因构建体的帮助下，将通过缺失和反义寡核苷酸策略分析参与异常剪接的顺式元件。将通过研究CLL和正常B细胞中差异表达的剪接因子来鉴定参与异常剪接的反式因子或剪接因子。这些剪接因子的作用将在小基因实验中通过表达或失活这些因子并确定它们对异常剪接的影响来证实。我们的目标是确定相关的顺式元件和结合它们的反式因子，以便尝试修改异常剪接过程和恢复E-钙粘蛋白表达。 公共卫生关系： E-钙粘蛋白基因的异常剪接。我们正在研究西方世界最常见的白血病，慢性淋巴细胞白血病（CLL）。这种白血病的发病率随着年龄的增长而增加，男性发病率较高。由于这些原因，CLL是退伍军人患者人群中的常见诊断。我们预测CLL患者结局的能力仍然有限，因此，我们无法为CLL患者量身定制特定治疗。识别CLL中新的遗传异常将有助于确定预后并开辟新的治疗途径。我们已经在CLL中发现了一种名为E-cadherin的基因的遗传异常，这是其他类型癌症中众所周知的基因。我们已经进行了大量的实验工作，这支持了我们的论点，E-钙粘蛋白是一个重要的基因在CLL。我们的建议将进一步研究该基因在CLL中的作用。