miRNA-Signaturen in Blutzellen von Lungentumorpatienten unter besonderer Berücksichtigung verschiedener Blutzellfraktionen

在我的第一次DFG资助期间，我能够向肺癌患者展示具有共同祖细胞(髓系或淋巴系祖细胞)的细胞，以及鼻尖细胞和适应性免疫系统的细胞在其miRNA表达方面显示出相似之处。5个白细胞亚群(T细胞、B细胞、NK细胞、嗜酸性粒细胞和中性粒细胞、单核细胞)的miRNA表达模式与全血不同。因此，不可能将整个血型追溯到其中一个白细胞亚群。这表明血液中的其他成分，主要是外切体，对全血miRNA的表达模式有影响。这一点的证据已经在最近的出版物中得到了证明。在我以前工作的基础上，我将在拟议的研究中分析肺癌患者和健康对照外显体中的miRNA模式。外体miRNA模式将与上述五个白细胞亚群的miRNA模式相关。为此，我将应用微阵列分析，就像我在第一次DFG拨款中所做的那样。利用miRNA微阵列数据，我将尝试推断患者和对照组miRNA模式中的哪些差异是由外切体或其中一个白细胞亚群造成的。此外，在一个更有功能的实验中，我将检测肿瘤外体和外体miRNAs对白细胞miRNA模式的影响。为此，健康捐赠者的白细胞将与不同的肺癌细胞系或从这些细胞系分离的外切体培养，并通过微阵列确定肿瘤外切体的miRNAs是否以及在多大程度上对白细胞的miRNome产生影响。此外，还将确定从肺癌患者血清中分离的外体miRNAs对健康供者白细胞miRNome的影响程度。为此，我将从肺癌患者的血清中分离外切体，并将其与健康捐赠者的白细胞孵育。然后比较外切体和白细胞的miRNA表达谱。为了确定外体或外体miRNAs是否以及在多大程度上对肿瘤的发生有任何直接影响，我将应用内皮管形成试验。在这里，肺癌患者和健康捐赠者的血清外切体将与内皮细胞孵育，以确定新血管的数量。内皮管形成试验的结果将与外体miRNome微阵列数据相关联，以确定对新血管形成贡献最大的miRNAs。通过这个拟议的项目，我将有助于理解外体miRNAs在肺癌中的作用。

During my first DFG grant I was able to show for lung cancer that cells with common progenitors (myeloide or lymphoide progenitors) and cells of the inate and the adaptive immune system show similarities in their miRNA expression. The miRNA expression pattern of five investigated leukocyte subpopulations (T cells, B cells, NK cells, eosinophile and neutrophile granulocytes, monocytes) were different compared to those of whole blood. Thus, it was not possible to trace the whole blood pattern back to one of the leukocyte subpopulations. This suggests that additional constituents in the blood, mainly exosomes, have an impact on the whole blood miRNA expression pattern. Evidence for this was already shown in recent publications. Based on my previous work, in the proposed study I will analyze miRNA pattern in exosomes of lung cancer patients and healthy controls. The exosomal miRNA pattern will be related to the miRNA pattern of the five leukocyte subpopulations mentioned above. To this end I will apply microarray analysis, as already done in my first DFG grant. With the miRNA microarray data I will try to deduce which differences in the miRNA pattern of patients and controls result from exosomes or one of the leukocyte subpopulations. In addition, in a more functional assay I will examine the influence of tumor exosomes and exosomal miRNAs on the miRNA pattern of leukocytes. To this end, leukocytes of a healthy donor will be cultivated with different lung cancer cell lines or exosomes isolated from those cell lines and with microarrays it will be determined if and to what extend miRNAs of the tumor exosomes have an effect on the miRNome of leukocytes. Additionally it will be determined to what extend exosomal miRNAs isolated from lung canecr patient serum have any influence on the miRNome of leukocytes of a healthy donor. To this end, i will isolate exosomes from serum of lung cancer patients and incubate these with leukocytes of a healthy donor. Then the miRNA expression profiles of exosomes and leukocytes will be compared. To determine if and to what extend exosomes or exosomal miRNAs have any direct impact on the tumorigenesis I will apply an endothelial tube formation assay. Here, exosomes of serum of lung cancer patients and of healthy donors will be incubated with endothelial cells to determine the number of new blood vessels. The results of the endothelial tube formation assay will be related to the exosomal miRNome microarray data to identfy miRNAs that contribute most to the fromation of new blood vessels. With the proposed project I will contribute to the understanding of the role of exosomal miRNAs in lung cancer.