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源于染色体异常，但其致病机制尚不清楚。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异常，很可能是通过代偿性替代NHEJ(ANHEJ)。ANHEJ在DSB修复连接处产生微同源性，但其成分仍然难以捉摸。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(S)可能抑制cNHEJ，从而通过aNHEJ促进染色体异常。目的1.抑制cNHEJ途径的miRNAs的鉴定。与Moskwa等人类似，我们检测到6个miRNAs候选。为了消除那些不影响cNHEJ基因的miRNAs，所有候选基因都将首先通过功能基因组整合的cNHEJ报告试验和荧光素酶试验进行预先测试。MIR-142已经进行了测试，结果令人振奋。随后，我们将测量这些miRNAs对DNA修复能力和对伽马电离辐射(GIR)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miRNAs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异常的DNA修复连接的微同源性以及miRNAs及其靶蛋白的表达。这样，我们将建立miRNAs与其蛋白质靶标的负相关以及与微同源的正相关。目的2.鉴定aNHEJ途径的新成分。我们将利用KrasG12D在胰腺细胞(小鼠模型)中的特异性表达和shRNA介导的Ku70(cNHEJ的核心成分)在体外对分离的腺泡细胞的下调来诱导aNHEJ的活性增加。所有在微阵列上上调的基因，在这两种情况下都是共同的，将被视为候选基因。基于文献检索、基因本体论和对前20个基因的排序，我们将建立一个最大基因库。50个基因。这些基因将使用siRNA在关注基因组aNHEJ报告的细胞系中被敲除，以消除所有候选基因，而不会对aNHEJ产生影响。我们还将确定这些蛋白质对DNA修复能力的影响和对GIR的动力学影响，以确定作用最强的前3个候选蛋白质。接下来，我们将操纵这些候选基因的表达，并测试它们对GIR、博莱霉素和PARP抑制剂的敏感性。为了体内相关性，我们将分析这些候选基因在KrasG12D/P48小鼠胰腺癌模型和人胰腺癌中的表达。为了确定其治疗价值，将候选基因特异性siRNA偶联到纳米颗粒上，用于治疗小鼠胰腺癌模型，并评价它们单独或与博莱霉素或PARP抑制剂联合应用对肿瘤生长/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.