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

所有癌症的一个特点是染色体畸变，但其致病机制尚不清楚。经典非同源末端连接（non-homologous end joining, cNHEJ）是DNA双链断裂（DSB）修复的关键机制之一，其功能障碍可能通过代偿性替代NHEJ （aNHEJ）引起染色体畸变。aNHEJ在DSB修复连接处产生微同源性，但其组分仍然难以捉摸。单个miRNA具有同时靶向单一或多种途径的多个基因的能力。miRNA(s)的异常表达可能抑制cNHEJ，从而通过aNHEJ导致染色体畸变。目的1。抑制cNHEJ通路的mirna的鉴定。与Moskwa等人类似，我们检测到6个候选mirna。为了消除那些不影响cNHEJ基因的mirna，所有候选人将首先使用功能基因组整合的cNHEJ报告基因测定和荧光素酶测定进行预测试。MiR-142已经进行了测试，并显示出良好的结果。随后，我们将测量这些miRNAs对DNA修复能力和伽玛电离辐射（gIR）下的动力学的影响，并确定过表达这些miRNAs的细胞对gIR、PARP抑制剂和博莱霉素的敏感性。为了支持我们的mirna在体内的意义，我们将分析胰腺细胞系和原发肿瘤组织中染色体畸变的DNA修复连接的微同源性以及mirna及其靶蛋白的表达。通过这种方式，我们将建立mirna与其蛋白靶点的负相关关系以及与微同源性的正相关关系。目标2。aNHEJ通路新组分的鉴定。我们将在胰腺细胞（小鼠模型）中特异性表达KrasG12D，并在分离的腺瘤细胞中通过shrna介导的Ku70 （cNHEJ的核心成分）的下调诱导aNHEJ活性的增加。微阵列上的所有上调基因，在这两种情况下都是常见的，将被视为候选基因。在文献检索、The Gene Ontology和排序前20个基因的基础上，我们将建立一个max。50个基因。这些基因将在含有基因组aNHEJ报告基因的细胞系中使用siRNA敲除，以消除所有对aNHEJ没有影响的候选基因。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，以确定影响最强的前3个候选蛋白。接下来，将对这些候选基因的表达进行调控，并检测其对gIR、博莱霉素和PARP抑制剂的敏感性。在体内相关性方面，我们将分析候选基因在KrasG12D/p48小鼠胰腺癌模型和人胰腺癌中的表达。为了确定治疗价值，候选基因特异性siRNA偶联纳米颗粒将用于治疗小鼠模型中的胰腺癌，并评估其单独或与博莱霉素或PARP抑制剂联合对肿瘤生长/转移的影响。

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.