Increased activity of alternative non-homologous end joining (A-NHEJ) as a possible mechanism of chromosomal aberration in pancreatic cancer.

选择性非同源末端连接 (A-NHEJ) 活性的增加是胰腺癌染色体畸变的可能机制。

• 批准号: 269137599
• 负责人: Dr. Patryk Moskwa
• 金额: --
• 依托单位: Klinik und Poliklinik für Innere Medizin A
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/269137599?language=en
• 关键词: Increased; activity; alternative; non; homologous

A hallmark of all cancer roots in chromosomal aberrations but their causative mechanism remain unclear. The classical non-homologous end joining (cNHEJ) is one of the key mechanisms of DNA double-strand brake (DSB) repair and its dysfunction causes chromosomal aberrations, most likely through the compensatory alternative NHEJ (aNHEJ). aNHEJ generates microhomology at the DSB repair junction whereas its components remain elusive.Single miRNA has the ability to target multiple genes of a single or multiple pathways simultaneously. Aberrant expression of miRNA(s) may supress cNHEJ contributing thereby via the aNHEJ to chromosomal aberrations.Aim 1. Identification of miRNAs suppressing the cNHEJ pathway. In analogy to in Moskwa et al., we detected 6 miRNAs candidates. To eliminate those miRNAs which don't impact cNHEJ genes, all candidates will first be pretested with a functional genome-integrated cNHEJ reporter-assay and with luciferase assay. MiR-142 was already tested and showed promising results. Subsequently, we will measure the impact of these miRNAs on DNA repair capacity and kinetic upon gamma-ionizing radiation (gIR) and determine the cells' sensitivity overexpressing these miRNAs to gIR, PARP inhibitor and Bleomycin. To support in vivo significance of our miRNAs, we will analyze the DNA repair junction of chromosomal aberration in pancreatic cell lines and primary tumor tissue for microhomology as well as expression of miRNAs and their target proteins. This way, we will establish an inverse correlation of miRNAs and their protein targets as well as positive correlation with microhomology.Aim 2. Identification of new components of the aNHEJ pathway. We will induce an increased activity of aNHEJ employing specific expression of KrasG12D in pancreatic cells (mouse model) and shRNA-mediated downregulation of Ku70 (a core component of cNHEJ) ex vivo in isolated acinus cells. All upregulated genes on microarrays, common to both conditions, will be considered as candidates. Based on literature search, The Gene Ontology and prioritizing the top 20 genes, we will establish a pool of max. 50 genes. These genes will be knocked down using siRNA in a cell line caring the genomic aNHEJ reporter to eliminate all candidates with no affect on aNHEJ. We will also determine the impact of these proteins on DNA repair capacity and kinetic upon gIR to identify the top 3 candidates with strongest effect. Next, the expression of these candidates will be manipulated and the sensitivity toward gIR, Bleomycin and PARP inhibitors tested.For in vivo relevance, the expression of the candidates in KrasG12D/p48 murine pancreatic cancer model and in human pancreatic cancer will be analyzed. To determine the therapeutic value, candidategenes specific siRNA coupled to nanoparticles will be used to treat pancreatic cancer in the murine model and their impact alone or in combination with Bleomycin or PARP inhibitor on tumor growth/metastasis evaluated.

所有癌症的一个标志是染色体畸变，但其致病机制仍不清楚。经典的非同源末端连接（cNHEJ）是DNA双链制动（DSB）修复的关键机制之一，其功能障碍导致染色体畸变，很可能通过代偿性替代NHEJ（aNHEJ）实现。aNHEJ在DSB修复连接处产生微同源性，而其组分仍然难以捉摸。单个miRNA具有同时靶向单个或多个途径的多个基因的能力。miRNA的异常表达可以抑制cNHEJ，从而经由aNHEJ促成染色体畸变。抑制cNHEJ途径的miRNA的鉴定。类似于Moskwa等人，我们检测到6个候选miRNAs。为了消除那些不影响cNHEJ基因的miRNA，所有候选物将首先用功能基因组整合的cNHEJ荧光素酶测定和荧光素酶测定进行预测试。MiR-142已经进行了测试，并显示出有希望的结果。随后，我们将测量这些miRNAs对DNA修复能力和动力学对γ-电离辐射（gIR）的影响，并确定过表达这些miRNAs的细胞对gIR、PARP抑制剂和博来霉素的敏感性。为了支持我们的miRNAs在体内的意义，我们将分析胰腺细胞系和原发性肿瘤组织中染色体畸变的DNA修复连接的微同源性以及miRNAs及其靶蛋白的表达。通过这种方式，我们将建立一个负相关的miRNAs和他们的蛋白质目标，以及正相关的微同源性。aNHEJ途径的新组分的鉴定。我们将采用KrasG 12 D在胰腺细胞中的特异性表达（小鼠模型）和在分离的腺泡细胞中离体的Ku 70（cNHEJ的核心组分）的shRNA介导的下调来诱导aNHEJ的活性增加。两种情况下共同的微阵列上的所有上调基因都将被视为候选基因。基于文献检索，基因本体论和优先排序的前20个基因，我们将建立一个池的最大。50个基因。这些基因将在携带基因组aNHEJ报告基因的细胞系中使用siRNA敲低，以消除所有候选物，而对aNHEJ没有影响。我们还将确定这些蛋白质对DNA修复能力和gIR动力学的影响，以确定具有最强效果的前3个候选物。接下来，将操纵这些候选物的表达并测试对gIR、博来霉素和PARP抑制剂的敏感性。对于体内相关性，将分析候选物在KrasG 12 D/p48鼠胰腺癌模型和人胰腺癌中的表达。为了确定治疗价值，将使用偶联至纳米颗粒的候选基因特异性siRNA来治疗鼠模型中的胰腺癌，并评估它们单独或与博来霉素或PARP抑制剂组合对肿瘤生长/转移的影响。