【摘要】：工作目的：DEK是一種在多細胞生物與部分單細胞生物中廣泛表達的癌蛋白,在大多數惡性腫瘤中高表達,而在終末分化的細胞中保持低表達水平。DEK可通過改變染色質結構,修復DNA,參與mRNA剪接與信號通路,或作為轉錄因子等途徑調控細胞的增殖、分化、凋亡、遷移、衰老等,與腫瘤細胞的形成有著重要的關系。然而關于DEK在肺腫瘤組織中的表達情況及其作用機理尚未有完善的研究成果。本研究將對肺癌細胞中DEK的表達量、作用、分子機制與轉錄調控進行檢測與探究,深化對DEK與腫瘤形成之間關系的了解。 研究方法：本實驗通過逆轉錄PCR (RT-PCR)與免疫組織化學實驗(IHC)檢測肺癌患者組織中DEK表達水平差異。并通過使用siRNA與DEK過表達載體轉染A549肺腺癌細胞,檢測DEK沉默與過表達的細胞功能與相關基因表達水平的改變。其中通過MTT法與集落形成法檢測DEK表達水平與細胞增殖能力之間的關系,Annexin V-PE細胞凋亡檢測試劑盒檢測DEK表達水平與細胞凋亡的關系、Matrigel細胞侵襲實驗檢測DEK表達水平與細胞侵襲能力之間的關系。此外,通過對肺癌患者基因組DNA進行DEK啟動子區(qū)域的BSP實驗,檢測其甲基化水平的差異,探究DEK啟動子區(qū)域甲基化水平與其轉錄調控的關系。 成果和結論：通過RT-PCR實驗發(fā)現,相對于肺正常組織,DEK mRNA在51.7%的肺腫瘤組織中發(fā)生高表達；其中有87.5%的肺腺癌組織發(fā)生DEK mRNA的高表達,高于其他組織學類型。而通過IHC實驗發(fā)現,肺腫瘤組織中,81.5%表現出DEK蛋白的陽性表達,39.5%表現出DEK蛋白的強陽性表達,強陽性率高于正常組織。進一步,通過使用siRNA與DEK過表達載體轉染A549肺腺癌細胞系,改變DEK表達量,檢測得到肺癌細胞DEK表達水平與細胞增殖能力和侵襲能力有著正相關關系,而與細胞凋亡率有著負相關關系,且p53、p65、ATM三種與DEK功能相關的基因表達量均與DEK的表達水平呈負相關關系。實驗同時發(fā)現,在肺腫瘤組織中,DEK啟動子區(qū)域CpG島的總甲基化水平與E2F和YY1等轉錄因子結合位點CpG的甲基化水平均有所下降。以上結果顯示,DEK在肺癌中的高表達與其作為癌基因的功能密切相關,其作用涉及p53、p65、ATM等DEK相關基因的作用,同時還提示DEK啟動子區(qū)域去甲基化與其轉錄活化及腫瘤形成之間的關系。
[Abstract]:Objective: DEK is a kind of oncoprotein widely expressed in multicellular organisms and some unicellular organisms. It is highly expressed in most malignant tumors and remains low in terminal differentiated cells. DEK can change chromatin structure. Repair DNA, is involved in mRNA splicing and signaling pathway, or as a transcription factor to regulate cell proliferation, differentiation, apoptosis, migration, aging, and has an important relationship with tumor cell formation. However, the expression and mechanism of DEK in lung neoplasms have not been well studied. In this study, the expression, role, molecular mechanism and transcriptional regulation of DEK in lung cancer cells were examined and explored to deepen the understanding of the relationship between DEK and tumor formation. Methods: reverse transcription PCR (RT-PCR) and immunohistochemical (IHC) were used to detect the expression of DEK in lung cancer. SiRNA and DEK overexpression vectors were used to transfect A549 lung adenocarcinoma cells to detect the changes of DEK silencing and overexpression cell function and the expression level of related genes. Among them, the relationship between DEK expression and cell proliferation was detected by MTT and colony forming assay, Annexin V-PE cell apoptosis detection kit was used to detect the relationship between DEK expression and cell apoptosis, and Matrigel cell invasion assay was used to detect DEK expression water. The relationship between flatness and cell invasiveness. In addition, the difference of methylation level in genomic DNA of lung cancer patients was detected by BSP test of DEK promoter region, and the relationship between methylation level of DEK promoter region and its transcriptional regulation was explored. Results and conclusions: compared with normal lung tissues, the expression of, DEK mRNA was higher in 51.7% of lung tumor tissues, and 87.5% of lung adenocarcinoma tissues had higher expression of DEK mRNA than other histological types. IHC assay showed that 81.5% of lung tumors showed positive expression of DEK protein, 39.5% showed strong positive expression of DEK protein, and the strong positive rate was higher than that of normal tissue. Furthermore, by using siRNA and DEK overexpression vector to transfect A549 lung adenocarcinoma cell line, the expression of DEK was changed, and the expression level of DEK in lung cancer cell line was positively correlated with cell proliferation and invasion ability. However, there was a negative correlation between the expression of p53 and p65ATM genes related to DEK function and the level of DEK expression. It was also found that the total methylation level of CpG island in the DEK promoter region and the CpG methylation level of transcription factor binding sites such as E2F and YY1 decreased in lung tumor tissues. These results suggest that the high expression of DEK in lung cancer is closely related to its function as a oncogene, and its role is related to the role of DEK related genes such as p53, p65, ATM, and so on. The relationship between demethylation of DEK promoter region and its transcriptional activation and tumor formation was also suggested.
【學位授予單位】：北京交通大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R734.2

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1 王璇;黃紹輝;於麗喬;劉潔;刁堯;孫長伏;;DEK原癌基因與口腔鱗狀細胞癌的關系[J];上海口腔醫(yī)學;2014年01期

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本文編號：2274655