人肺腺癌细胞A549中DEK的作用机制研究
发布时间：2018-07-15 21:02
【摘要】：目的:DEK是广泛存在于细胞核内高度保守的可磷酸化癌蛋白,在人类许多恶性肿瘤中均出现过表达,表现出与肿瘤的发生发展密切相关。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。目前关于DEK促进肿瘤发展的研究越来越多,然而关于DEK与肺癌的关系以及DEK在肺癌中的作用机制尚未有完善的研究成果。前期研究结果显示,DEK蛋白在人肺癌肿瘤组织中的表达量明显高于正常组织,提示DEK可能在肺癌的发病机制中发挥重要作用,因此本研究在前期工作的基础上,进一步在细胞层面上研究DEK沉默对肺癌细胞转录组的影响,并深入探讨DEK在肺癌中的作用机制,为肺癌的治疗提供新的途径与实验依据。方法:本实验通过实时荧光定量RT-PCR与Western Blotting检测6种肺癌细胞系中DEK表达水平差异,从中筛选出内源性高水平表达DEK的细胞,并通过RNAi慢病毒载体构建DEK沉默的A549细胞,通过RNA-Seq检测由DEK沉默诱导的差异表达基因,再通过GO分析与Pathway分析评估差异表达基因与肺癌的关系,最后在细胞层面上验证分析的准确性,通过MTT法检测DEK沉默对A549细胞增殖能力的影响,PI染色法检测DEK沉默对A549细胞周期的影响,Annexin V-FITC/PI细胞凋亡检测试剂盒检测DEK沉默对A549细胞凋亡的影响,此外,通过实时荧光定量RT-PCR与Western Blotting检测IGFBP3/IGF-1R/AKT通路相关基因的表达水平。结果:通过GO分析与Pathway分析评估DEK沉默的A549细胞中差异表达基因与肺癌的关系,结果显示DEK与细胞生长和死亡密切相关。进一步,使用DEK siRNA转染A549细胞,检测DEK沉默对A549细胞功能的影响,结果显示DEK沉默可抑制A549细胞的增殖,引起A549细胞周期在G1期发生阻滞,而且能够显著促进细胞的凋亡。最后检测IGFBP3/IGF-1R/AKT通路相关基因表达量与DEK表达水平的关系,结果显示,IGFBP3基因表达量与DEK的表达水平呈负相关关系,IGF-1R、p-AKT、AKT基因表达量与DEK的表达水平呈正相关关系。结论:综上实验结果我们得出结论,DEK沉默可以引起A549细胞周期G1期阻滞,抑制A549细胞的增殖,并能显著促进细胞的凋亡,且其分子机制与IGFBP3/IGF-1R/AKT 通路相关。
[Abstract]:Objective: Dek is a highly conserved phosphorylated oncoprotein widely present in the nucleus. It has been expressed in many human malignant tumors, showing that it is closely related to the development of tumor. DEK can change the chromatin structure by changing the chromatin structure. Repairing DNA, participating in mRNA splicing and signaling pathway, or regulating cell proliferation, differentiation, apoptosis, migration, senescence and other pathways as transcription factors play an important role in the formation of tumor cells. At present, there are more and more researches on DEK promoting tumor development. However, the relationship between DEK and lung cancer and the mechanism of DEK in lung cancer have not been well studied. Previous studies showed that the expression of dek protein in human lung cancer tissues was significantly higher than that in normal tissues, suggesting that DEK may play an important role in the pathogenesis of lung cancer. The effect of DEK silencing on the transcriptome of lung cancer cells was further studied at the cellular level, and the mechanism of DEK in lung cancer was discussed in order to provide a new approach and experimental basis for the treatment of lung cancer. Methods: the expression levels of DEK in 6 lung cancer cell lines were detected by real-time fluorescent quantitative RT-PCR and Western blotting. The cells with high level of DEK expression were screened out, and the A549 cells were silenced by RNAi lentivirus vector. The differentially expressed genes induced by DEK silencing were detected by RNA-Seq, and the relationship between differentially expressed genes and lung cancer was evaluated by go analysis and Pathway analysis. The effect of DEK silencing on the proliferation of A549 cells was detected by MTT assay. The effect of DEK silencing on A549 cell cycle was detected by Pi staining. The effect of dek silencing on A549 cell apoptosis was detected by Annexin V-FITC / Pi cell apoptosis assay kit. The expression of IGFBP3 / IGF-1R / AKT related genes was detected by real-time quantitative RT-PCR and Western blotting. Results: go analysis and Pathway analysis were used to evaluate the relationship between differentially expressed genes and lung cancer in A549 cells silenced by DEK. The results showed that DEK was closely related to cell growth and death. Furthermore, DEK siRNA was used to transfect A549 cells, and the effect of DEK silencing on A549 cell function was detected. The results showed that DEK silencing could inhibit the proliferation of A549 cells, induce cell cycle arrest in G1 phase, and significantly promote the apoptosis of A549 cells. Finally, the relationship between the expression of IGFBP3 / IGF-1R / AKT pathway and the expression level of DEK was detected. The results showed that the expression of IGFBP3 gene was negatively correlated with the expression level of DEK. There was a positive correlation between the expression of IGFBP3 gene and the expression level of DEK. Conclusion: in conclusion, the silencing of A549 cells can induce G1 phase arrest, inhibit the proliferation of A549 cells, and promote the apoptosis of A549 cells, and its molecular mechanism is related to the IGFBP3 / IGF-1R / AKT pathway.
【学位授予单位】：北京交通大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
，
本文编号：2125371
[Abstract]:Objective: Dek is a highly conserved phosphorylated oncoprotein widely present in the nucleus. It has been expressed in many human malignant tumors, showing that it is closely related to the development of tumor. DEK can change the chromatin structure by changing the chromatin structure. Repairing DNA, participating in mRNA splicing and signaling pathway, or regulating cell proliferation, differentiation, apoptosis, migration, senescence and other pathways as transcription factors play an important role in the formation of tumor cells. At present, there are more and more researches on DEK promoting tumor development. However, the relationship between DEK and lung cancer and the mechanism of DEK in lung cancer have not been well studied. Previous studies showed that the expression of dek protein in human lung cancer tissues was significantly higher than that in normal tissues, suggesting that DEK may play an important role in the pathogenesis of lung cancer. The effect of DEK silencing on the transcriptome of lung cancer cells was further studied at the cellular level, and the mechanism of DEK in lung cancer was discussed in order to provide a new approach and experimental basis for the treatment of lung cancer. Methods: the expression levels of DEK in 6 lung cancer cell lines were detected by real-time fluorescent quantitative RT-PCR and Western blotting. The cells with high level of DEK expression were screened out, and the A549 cells were silenced by RNAi lentivirus vector. The differentially expressed genes induced by DEK silencing were detected by RNA-Seq, and the relationship between differentially expressed genes and lung cancer was evaluated by go analysis and Pathway analysis. The effect of DEK silencing on the proliferation of A549 cells was detected by MTT assay. The effect of DEK silencing on A549 cell cycle was detected by Pi staining. The effect of dek silencing on A549 cell apoptosis was detected by Annexin V-FITC / Pi cell apoptosis assay kit. The expression of IGFBP3 / IGF-1R / AKT related genes was detected by real-time quantitative RT-PCR and Western blotting. Results: go analysis and Pathway analysis were used to evaluate the relationship between differentially expressed genes and lung cancer in A549 cells silenced by DEK. The results showed that DEK was closely related to cell growth and death. Furthermore, DEK siRNA was used to transfect A549 cells, and the effect of DEK silencing on A549 cell function was detected. The results showed that DEK silencing could inhibit the proliferation of A549 cells, induce cell cycle arrest in G1 phase, and significantly promote the apoptosis of A549 cells. Finally, the relationship between the expression of IGFBP3 / IGF-1R / AKT pathway and the expression level of DEK was detected. The results showed that the expression of IGFBP3 gene was negatively correlated with the expression level of DEK. There was a positive correlation between the expression of IGFBP3 gene and the expression level of DEK. Conclusion: in conclusion, the silencing of A549 cells can induce G1 phase arrest, inhibit the proliferation of A549 cells, and promote the apoptosis of A549 cells, and its molecular mechanism is related to the IGFBP3 / IGF-1R / AKT pathway.
【学位授予单位】：北京交通大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
，
本文编号：2125371
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