肺癌特异性DKK1基因转录调控元件的分析预测及活性筛选-生物化学与分子生物学专业论文.docxVIP

Abstra Abstract 华侨大学硕士学位论文 华侨大学硕士学位论文 万方数据 万方数据 万方数据 万方数据 Abstract DKK1 is known as an antagonist of the canonical Wnt signalling pathway. It can block canonical Wnt signaling by forming a ternary complex with Lrps. Also, it can combine with Kremen and Lrp6 transmembrane receptor. Thereby, a rapid endocytosis and removal of the Lrp receptor from the plasmamembrane is induced. A specificity and high level expression of DKK1 is observed in lung cancer. DKK1 is hardly expressed in normal human adult tissues, except in embryonic tissues. But it have been also shown that high levels of DKK1 could be speci?cally measured in multiple lung cancer cell lines and lung cancer tissues. DKK1 concentrations were significantly higher in patients with lung cancer than in pa-tients with other malignant tumors or benign lung diseases and healthy controls. The use of both DKK1 and other biomark could increase sensitivity to detect lung cancer. Our study, however, found that activity of DKK1 promoter in lung cancer cell was low. This may indictate that there were some enhancer elements having important roles in expression regulation of DKK1. The key point of this study is how to predict and locate the position of transcriptional regulatory elements. Here we cloned four different segments of DKK1 promoter into pGL3-basic plasmid. Then we transferred these recombinant plasmids into A549、H460、H446、Eca-109、Chang liver cells and identified DKK1-5 as proximal promoter though Dual-Luciferase Reporter Assay System. We then had free access to information of DNaseI hypersensitive sites and histone modification in UCSC genome browser from which, we obtained 12 candidate DNA sequences whose functions might act as an enhancer. We then insected them into upstream of DKK1 promoter and tested their biological activity through Dual-Luciferase Reporter Assay System. Finally six candidate sequences, i.e. TRE3、TRE4、TRE6、TRE7、TRE8 and TRE12, were obtained. They were of significantly higher activity of DKK