DEK在肺癌细胞中的表达水平及其作用机理的研究
发布时间：2018-10-16 14:27
【摘要】：工作目的：DEK是一种在多细胞生物与部分单细胞生物中广泛表达的癌蛋白,在大多数恶性肿瘤中高表达,而在终末分化的细胞中保持低表达水平。DEK可通过改变染色质结构,修复DNA,参与mRNA剪接与信号通路,或作为转录因子等途径调控细胞的增殖、分化、凋亡、迁移、衰老等,与肿瘤细胞的形成有着重要的关系。然而关于DEK在肺肿瘤组织中的表达情况及其作用机理尚未有完善的研究成果。本研究将对肺癌细胞中DEK的表达量、作用、分子机制与转录调控进行检测与探究,深化对DEK与肿瘤形成之间关系的了解。 研究方法：本实验通过逆转录PCR (RT-PCR)与免疫组织化学实验(IHC)检测肺癌患者组织中DEK表达水平差异。并通过使用siRNA与DEK过表达载体转染A549肺腺癌细胞,检测DEK沉默与过表达的细胞功能与相关基因表达水平的改变。其中通过MTT法与集落形成法检测DEK表达水平与细胞增殖能力之间的关系,Annexin V-PE细胞凋亡检测试剂盒检测DEK表达水平与细胞凋亡的关系、Matrigel细胞侵袭实验检测DEK表达水平与细胞侵袭能力之间的关系。此外,通过对肺癌患者基因组DNA进行DEK启动子区域的BSP实验,检测其甲基化水平的差异,探究DEK启动子区域甲基化水平与其转录调控的关系。 成果和结论：通过RT-PCR实验发现,相对于肺正常组织,DEK mRNA在51.7%的肺肿瘤组织中发生高表达；其中有87.5%的肺腺癌组织发生DEK mRNA的高表达,高于其他组织学类型。而通过IHC实验发现,肺肿瘤组织中,81.5%表现出DEK蛋白的阳性表达,39.5%表现出DEK蛋白的强阳性表达,强阳性率高于正常组织。进一步,通过使用siRNA与DEK过表达载体转染A549肺腺癌细胞系,改变DEK表达量,检测得到肺癌细胞DEK表达水平与细胞增殖能力和侵袭能力有着正相关关系,而与细胞凋亡率有着负相关关系,且p53、p65、ATM三种与DEK功能相关的基因表达量均与DEK的表达水平呈负相关关系。实验同时发现,在肺肿瘤组织中,DEK启动子区域CpG岛的总甲基化水平与E2F和YY1等转录因子结合位点CpG的甲基化水平均有所下降。以上结果显示,DEK在肺癌中的高表达与其作为癌基因的功能密切相关,其作用涉及p53、p65、ATM等DEK相关基因的作用,同时还提示DEK启动子区域去甲基化与其转录活化及肿瘤形成之间的关系。
[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.
【学位授予单位】：北京交通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2
本文编号：2274655
[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.
【学位授予单位】：北京交通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2
【参考文献】
相关期刊论文 前1条
1 王璇;黄绍辉;於丽乔;刘洁;刁尧;孙长伏;;DEK原癌基因与口腔鳞状细胞癌的关系[J];上海口腔医学;2014年01期
，本文编号：2274655
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2274655.html
