延伸复合体3（ELP3）对肺癌细胞A549的作用及调节机制研究

发布时间：2018-04-13 16:24

本文选题：ELP3 + 肺癌细胞A549　；参考：《内蒙古大学》2015年硕士论文

【摘要】：DNA甲基化和组蛋白修饰等表观遗传调节对基因表达调控有重要作用。研究表明,肿瘤形成过程中,表观遗传修饰的改变导致基因表达异常,细胞发生癌变。延伸复合体3(ELP3)是一种与转录过程密切相关的组蛋白乙酰化转移酶,具有铁硫簇结构域(SAM),对DNA去甲基化具有重要作用,但ELP3是如何调节肺癌细胞的细胞特性和恶性表型的作用还不清楚。本研究检测了ELP3在肺癌细胞A549的表达定位,并通过基因敲减研究了ELP3对肺癌细胞的影响及作用机制。本研究首先利用实时定量PCR检测了不同肺癌细胞系中ELP3 mRNA水平的表达,结果表明,肺癌细胞A549、H460、SK-MES-1的ELP3 mRNA表达水平均高于肺上皮细胞,肺癌细胞系A549的表达水平最高,故选择A549做后续实验。其次,通过构建pGPU6/GFP/Neo siRNA表达载体,利用脂质体法转染A549细胞,经G418筛选,Real-time PCR检测表明转染细胞中ELP3 mRNA的表达与对照组相比降低了85.4%(p0.01),说明ELP3的表达受到抑制。之后,利用MTT、Traswell小室和软琼脂集落实验对ELP3敲减后癌细胞的恶性表型进行了检测。MTT结果表明,与对照细胞相比,ELP3敲减后细胞增殖能力下降了24.61%(p0.05)；Traswell小室和软琼脂集落结果表明,细胞迁移能力下降了52.38%(p0.01),侵袭能力下降了37.90%(p0.05)；软琼脂集落实验表明,锚定非依赖生长能力显著降低了83.57%(p0.01)。为进一步研究ELP3可能的作用机理,本研究检测了ELP3敲减后对相关基因表达及相应的基因启动子区表观修饰的影响。实时定量PCR结果表明,ELP3基因被敲减后,BCL2、C-erbA、C-JUN、C-myc、N-myc的mRNA表达显著下调,KLF4的mRNA的表达水平上调,K-ras、SOX2的转录水平未受影响。亚硫酸氢盐PCR测序结果显示,与对照组相比,BCL2启动子区DNA甲基化水平提高了5.00%, K-ras启动子区甲基化水平提高了8.46%。α卫星序列(alpha satellit e)甲基化水平降低了10%,微卫星序列32的长末端重复序列(retroviral LTR sequence of minisatellite 32)的甲基化水平提高了18.30%。ChIP研究结果显示,敲减ELP3基因后,C-myc和SOX2基因启动子区组蛋白修饰H3K9me3、H3K4me3、 H3K27me3水平显著降低。综上所述,ELP3在肺癌细胞中高表达,ELP3敲减后癌基因的表达受到抑制,相应的癌基因启动子区的DNA甲基化水平有所提高,另外,敲减ELP3基因后,相关基因启动子区组蛋白修饰也发生改变。表明ELP3在肺癌细胞具有DNA去甲基化作用,ELP3可能通过影响相关基因启动子区DNA甲基化和组蛋白修饰,改变基因表达水平,从而影响肺癌细胞的增殖、迁移、侵袭和锚定非依赖生长能力。
[Abstract]:Epigenetic regulation such as DNA methylation and histone modification plays an important role in the regulation of gene expression.The results showed that the epigenetic modification resulted in abnormal gene expression and carcinogenesis during tumor formation.Extension complex 3 (ELP3) is a histone acetyltransferase, which is closely related to transcription process, and has a ferrithione cluster domain, SAMN, which plays an important role in demethylation of DNA.But it is not clear how ELP3 regulates the cellular properties and malignant phenotypes of lung cancer cells.In this study, the expression of ELP3 in lung cancer cell line A549 was detected, and the effect of ELP3 on lung cancer cell line A549 was studied by gene knockdown.In this study, real-time quantitative PCR was used to detect the expression of ELP3 mRNA in different lung cancer cell lines. The results showed that the expression of ELP3 mRNA in lung cancer cell line A549 was higher than that in lung epithelial cell line, and the expression level of ELP3 mRNA in lung cancer cell line A549 was the highest.So choose A549 to do follow-up experiment.Secondly, the pGPU6/GFP/Neo siRNA expression vector was constructed and transfected into A549 cells by liposome method. The results of G418 screening and Real-time PCR detection showed that the expression of ELP3 mRNA in the transfected cells was lower than that in the control group by 85.4p0.01, indicating that the expression of ELP3 was inhibited.After that, the malignant phenotype of cancer cells after ELP3 knockout was detected by MTT taswell chamber and soft Agar colony assay. The results showed that compared with the control cells, the proliferative ability of the cells after ELP3 knockout was decreased by 24.61% Traswell chamber and soft Agar colony assay.The ability of cell migration and invasion decreased by 52.38% and 37.90%, respectively, and the ability of anchoring independent growth was significantly decreased by 83.57% (p0.01) in soft Agar colony assay.In order to further study the possible mechanism of ELP3, the effects of ELP3 knockout on the expression of related genes and the corresponding epidermal modification of gene promoter were investigated.The results of real-time quantitative PCR showed that the expression of C-erbAerbAC-JUNC-JUNC N-myc mRNA was significantly down-regulated after the knockout of ELP3 gene. The expression level of mRNA of KLF4 was up-regulated and the transcription level of K-rassox2 was not affected.The results of PCR sequencing showed that,Compared with the control group, the methylation level of DNA in the BCL2 promoter increased 5.00, the methylation level in the K-ras promoter increased 8.46%. The methylation level of 伪 -satellite sequence alpha satellit decreased by 10%, and the long terminal repeat sequence of microsatellite sequence 32 was retroviral LTR sequence of minisatellite.The methylation level of 32) increased the level of methylation in the 18.30%.ChIP study.After knockout of ELP3 gene, the level of H3K27me3 decreased significantly when the promoter protein of C-myc and SOX2 gene was modified with histone of H3K9me3m3C3K4me3.In conclusion, the overexpression of ELP3 in lung cancer cells was inhibited, and the DNA methylation level in the promoter region of the corresponding oncogene was increased. In addition, after knockout of the ELP3 gene,The histone modification of the promoter region of the related gene also changed.The results suggest that ELP3 has DNA demethylation effect in lung cancer cells. ELP3 may affect the proliferation and migration of lung cancer cells by affecting the DNA methylation and histone modification in the promoter region of lung cancer cells.Invasion and anchoring independent growth capacity.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S857.4

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