Aberrant splicing of the E-cadherin gene.

描述(由申请人提供)： 项目摘要：E-钙粘素基因的异常剪接。慢性淋巴细胞白血病(CLL)是最常见的白血病，也是一种不治之症。为了确定慢性淋巴细胞白血病细胞中新的基因突变，我们使用微阵列技术来筛选含有提前终止密码子(PTCs)的基因。这些PTC突变通过表达截短的非功能mRNA来促进肿瘤的发生，该mRNA被无义介导的降解(NMD)途径降解，从而导致表达缺失。在上调最高的基因中，我们发现了E-钙粘素基因，这是一种肿瘤抑制基因。PTC处于E-钙粘素转录本的选择性剪接中，当NMD途径被阻断时，该转录本稳定下来。E-钙粘蛋白的这一转录本完全缺乏外显子11(外显子跳跃)，导致外显子12发生移码和PTC密码子。RT-PCR分析表明，正常B细胞也存在外显子11跳跃，但频率要低得多。我们还发现，在62%的CLL标本(n=35)中，野生型E-cadherin总RNA的表达显著降低。此外，在慢性淋巴细胞性白血病标本中，高异常转录本和低E-钙粘附素表达之间存在负相关(p=0.018)。我们的结果提示了一种新的E-钙粘蛋白基因功能丧失的机制，即CLL细胞中的反式因子/剪接因子导致该肿瘤抑制基因的非生产性/异常剪接增加。E-钙粘附素缺失的一个下游效应是WNT途径的激活，因为β-连环素现在能够转移到细胞核。这一途径在CLL细胞中是活跃的，我们使用报告分析的数据证实了这一发现。我们的初步实验还表明，在E-钙粘附素表达较低的CLL标本中，报告活性较高，并且E-钙粘附素单独异位表达可以逆转报告活性。在特定的目标1中，我们将研究E-钙粘附素在CLL细胞中表达缺失的意义。E-钙粘附素的缺失可导致WNT-连环蛋白途径的激活，该途径也可被其他WNT途径激活剂(如WNT和卷曲基因)激活。我们将确定E-钙粘附素丢失和WNT/Frizzleed基因在WNT途径上调中的相对作用。实验将通过E-钙粘附素的重新表达及其对细胞凋亡、迁移和侵袭的影响来抑制这一途径。在特定的目标2中，将研究参与外显子11异常剪接的顺式和反式元件。在微型基因构建的帮助下，参与异常剪接的顺式元件将通过缺失和反义寡核苷酸策略进行分析。通过研究CLL和正常B细胞中差异表达的剪接因子，可以确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微型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-钙粘素的表达。

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.