MLL2促进肺癌A549细胞发生发展中的作用机制及萝卜籽素抑制肺癌A549细胞的生物信息学研究

发布时间：2018-06-24 18:32

本文选题：MLL2 + A549肺癌细胞　；参考：《南昌大学》2016年博士论文

【摘要】：目的:肺癌对人类健康和生活造成巨大危害,并且肺癌五年生存率不到15%,是人类因癌症死亡的主要原因。许多致癌物质都可以通过癌基因和抑癌基因的激活或抑制促使活细胞转化为癌症细胞。MLL2存在于各种上皮细胞和组织的癌细胞中,如前列腺癌,结肠癌,肺癌和肝癌中被发现。因为这些肿瘤细胞中MLL2基因启动子的Cp G岛的甲基化显著增加,因此它被认为是一种肿瘤基因。本研究主要针对MLL2对肺癌细胞的影响进行研究,讨论了MLL2基因诱导人肺腺癌A549细胞增殖,迁移和侵袭的可能机制,研究了肺癌MLL2基因对组蛋白H3和H4赖氨酸残基甲基化的影响,最后通过生物信息学方法分析了可能的相关基因与信号转导通路。方法:1、从构建过表达和基因沉默MLL2的A549肺癌细胞出发,研究这两种A549肺癌细胞的生物学行为的影响。2、通过脂质体转染含有MLL2基因的质粒,RT-PCR法,Western Blot,CCK-8细胞测定蛋白表达、以及A549肺癌细胞增殖和集落形成。3、采用荧光定量PCR(FQ-PCR)检测MLL2,MMP-2(基质金属蛋白酶-2),PKR(双链RNA依赖性蛋白激酶)的m RNA表达,Western Blot检测PKR、MLL2和MMP-2的表达;用Transwell小室进行迁移和侵袭实验检测A549细胞增殖。4、过表达和沉默MLL2质粒转染到人肺腺癌A549细胞,采用Western Blot和免疫荧光试验检测MLL2对H3K4me3,H3k4me2,H3k4me1,H3K9me3,H3k9me2,H3k9me1,H3k27me3,H3k27me2,H3k27me1,H3k36me3,H3k36me2,H3k36me1,H4k20me3的甲基化状态的作用。5、采用R/Bioconductor分析和挖掘基因芯片的数据。下载GEO中包含经过萝卜籽素处理前后的A549肺癌细胞的原始数据,然后进行标准化处理、质控、差异基因的筛选与分析、GO分析、聚类以及通路总结,进一步的分析基因调控网络以及分子相互作用。结果:我们成功构建了过表达MLL2的A549肺癌细胞,通过MTT法检测,发现该细胞过度表达MLL2基因,与空载体组和对照组相比,过表达MLL2的细胞侵袭迁移显著增加,同类细胞之间粘附降低,异质细胞粘附则增强了,而且扩散加速和集落形成率均发生了提高。此外,MLL2促使MMP-2 m RNA表达显著增加;P-PKR蛋白的表达则降低;PKR m RNA的表达与对照组和空载体组相比无显著差异。Western Blot分析表明,MLL2造成H3K9me3的表达削弱,而沉默MLL2增强H3K9me3的表达,H3组蛋白20位点和H4组蛋白4、27、36位点的甲基化状态则没有显著差异。免疫荧光表明MLL2造成H3K9me3表达增强,并且与Western Blot结果一致。最后,通过生物信息学的结果,我们发现了经过萝卜籽素处理前后A549肺癌细胞与MLL2相关的癌基因发生了下调。结论:我们发现过度表达MLL2可以促进A549肺癌细胞生长,促进MMP-2和PKR磷酸化,促进肺腺癌A549细胞增殖,迁移和侵袭。并且,肺癌基因MLL2有去H3K9me3甲基化的功能,而且没有对其它赖氨酸产生甲基化。最后,通过生物信息学的结果,我们发现萝卜籽素的处理可以抑制A549肺癌细胞MLL2相关的癌基因,有助于抑制肺癌细胞的发展。
[Abstract]:Objective: lung cancer does great harm to human health and life, and the 5-year survival rate of lung cancer is less than 15%, which is the main cause of human cancer death. Many carcinogens can be activated or suppressed by oncogenes and suppressor genes to induce the transformation of living cells into cancer cells. MLL2 is found in cancer cells of various epithelial cells and tissues such as prostate cancer colon cancer lung cancer and liver cancer. Because the methylation of the CP G island of the MLL2 gene promoter is significantly increased in these tumor cells, it is considered to be a tumor gene. In this study, the effect of MLL2 on lung cancer cell line was studied. The possible mechanism of MLL2 gene induced proliferation, migration and invasion of human lung adenocarcinoma A549 cell line was discussed, and the effect of MLL2 gene on histone H3 and H4 lysine residue methylation was studied. Finally, the related genes and signal transduction pathways were analyzed by bioinformatics. Methods: to investigate the biological behavior of A549 lung cancer cells by constructing overexpression and gene silencing MLL2 cells, the biological behavior of these two A549 lung cancer cells was studied. The expression of protein was detected by Western BlotCK-8 cells transfected with the plasmid containing MLL2 gene by RT-PCR. And the proliferation and colony formation of A549 lung cancer cells. The mRNA expression of MLL2mpMMP-2 (matrix metalloproteinase-2) and PKR (double-stranded RNA-dependent protein kinase) was detected by fluorescence quantitative PCR (FQ-PCR). Western blot was used to detect the expression of PKRMLL2 and MMP-2. The proliferation of A549 cells was detected by transwell chamber migration and invasion assay. Overexpression and silencing of MLL2 plasmid were transfected into human lung adenocarcinoma A549 cells. The effect of MLL2 on the methylation state of H3K4me3me3me2H3k4me1h3k9me1H3k9me1H3k27me3me2H3k27me2H3k27me2H3k36me2H3k36me2H3k36me2H3k36me2H4k20me3 was detected by Western Blot and immunofluorescence assay, and the data of H3k36me2H3k36me1H4k20me3 was analyzed and mined by RBioconductor. The GEO contains raw data of A549 lung cancer cells before and after treatment with radish seed hormone, then standardized treatment, quality control, screening and analysis of differential genes, clustering and pathway summarization. Further analysis of gene regulatory networks and molecular interactions. Results: A549 lung cancer cells expressing MLL2 were successfully constructed. The overexpression of MLL2 gene was detected by MTT assay. Compared with empty vector group and control group, overexpression of MLL2 increased significantly in invasion and migration of A549 lung cancer cells. The adhesion between the same cells decreased, the adhesion of heterogeneous cells increased, and the diffusion rate and colony formation rate increased. In addition, MLL2 increased the expression of MMP-2 mRNA significantly, but decreased the expression of pPKR mRNA. Western blot analysis showed that MLL2 induced the decrease of H3K9me3 expression. There was no significant difference in methylation between H3K9me3 histone 20 and H4 histone 42736 by silencing MLL2. Immunofluorescence showed that MLL 2 increased the expression of H 3K 9me3, which was consistent with the results of Western Blot. Finally, we found that the MLL2-related oncogene was down-regulated in A549 lung cancer cells before and after treatment with radish seed. Conclusion: overexpression of MLL2 can promote the growth of A549 lung cancer cells, promote the phosphorylation of MMP-2 and PKR, and promote the proliferation, migration and invasion of A549 cells. Moreover, the lung cancer gene MLL2 has the function of demethylation of H _ 3K _ 9me _ 3 and does not produce methylation of other lysine. Finally, through the bioinformatics results, we found that the treatment of radish seed can inhibit the MLL2-related oncogene in A549 lung cancer cell line, which is helpful to inhibit the development of lung cancer cells.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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