【摘要】：目的:DEK是廣泛存在于細(xì)胞核內(nèi)高度保守的可磷酸化癌蛋白,在人類許多惡性腫瘤中均出現(xiàn)過表達(dá),表現(xiàn)出與腫瘤的發(fā)生發(fā)展密切相關(guān)。DEK可通過改變?nèi)旧|(zhì)結(jié)構(gòu),修復(fù)DNA,參與mRNA剪接與信號通路,或作為轉(zhuǎn)錄因子等途徑調(diào)控細(xì)胞的增殖、分化、凋亡、遷移、衰老等,與腫瘤細(xì)胞的形成有著重要的關(guān)系。目前關(guān)于DEK促進(jìn)腫瘤發(fā)展的研究越來越多,然而關(guān)于DEK與肺癌的關(guān)系以及DEK在肺癌中的作用機(jī)制尚未有完善的研究成果。前期研究結(jié)果顯示,DEK蛋白在人肺癌腫瘤組織中的表達(dá)量明顯高于正常組織,提示DEK可能在肺癌的發(fā)病機(jī)制中發(fā)揮重要作用,因此本研究在前期工作的基礎(chǔ)上,進(jìn)一步在細(xì)胞層面上研究DEK沉默對肺癌細(xì)胞轉(zhuǎn)錄組的影響,并深入探討DEK在肺癌中的作用機(jī)制,為肺癌的治療提供新的途徑與實驗依據(jù)。方法:本實驗通過實時熒光定量RT-PCR與Western Blotting檢測6種肺癌細(xì)胞系中DEK表達(dá)水平差異,從中篩選出內(nèi)源性高水平表達(dá)DEK的細(xì)胞,并通過RNAi慢病毒載體構(gòu)建DEK沉默的A549細(xì)胞,通過RNA-Seq檢測由DEK沉默誘導(dǎo)的差異表達(dá)基因,再通過GO分析與Pathway分析評估差異表達(dá)基因與肺癌的關(guān)系,最后在細(xì)胞層面上驗證分析的準(zhǔn)確性,通過MTT法檢測DEK沉默對A549細(xì)胞增殖能力的影響,PI染色法檢測DEK沉默對A549細(xì)胞周期的影響,Annexin V-FITC/PI細(xì)胞凋亡檢測試劑盒檢測DEK沉默對A549細(xì)胞凋亡的影響,此外,通過實時熒光定量RT-PCR與Western Blotting檢測IGFBP3/IGF-1R/AKT通路相關(guān)基因的表達(dá)水平。結(jié)果:通過GO分析與Pathway分析評估DEK沉默的A549細(xì)胞中差異表達(dá)基因與肺癌的關(guān)系,結(jié)果顯示DEK與細(xì)胞生長和死亡密切相關(guān)。進(jìn)一步,使用DEK siRNA轉(zhuǎn)染A549細(xì)胞,檢測DEK沉默對A549細(xì)胞功能的影響,結(jié)果顯示DEK沉默可抑制A549細(xì)胞的增殖,引起A549細(xì)胞周期在G1期發(fā)生阻滯,而且能夠顯著促進(jìn)細(xì)胞的凋亡。最后檢測IGFBP3/IGF-1R/AKT通路相關(guān)基因表達(dá)量與DEK表達(dá)水平的關(guān)系,結(jié)果顯示,IGFBP3基因表達(dá)量與DEK的表達(dá)水平呈負(fù)相關(guān)關(guān)系,IGF-1R、p-AKT、AKT基因表達(dá)量與DEK的表達(dá)水平呈正相關(guān)關(guān)系。結(jié)論:綜上實驗結(jié)果我們得出結(jié)論,DEK沉默可以引起A549細(xì)胞周期G1期阻滯,抑制A549細(xì)胞的增殖,并能顯著促進(jìn)細(xì)胞的凋亡,且其分子機(jī)制與IGFBP3/IGF-1R/AKT 通路相關(guān)。
[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.
【學(xué)位授予單位】：北京交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R734.2
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本文編號：2125371