EGR-1对ASPP1基因调控机制的研究

发布时间：2018-06-27 23:48

  本文选题：槲皮素 + EGR1　； 参考：《哈尔滨工业大学》2016年硕士论文

【摘要】：ASPP1是新发现的蛋白家族ASPPs中的一个抑癌基因,其表达量在许多种类的癌症组织中下调,因此揭示其上游调控机制对解释其表达量下降具有重要的意义。目前已发现的主要是表观遗传水平的调控,而在转录水平上调控ASPP1基因的转录因子现已有报道的仅有E2F家族的成员。本文就EGR-1对ASPP1的调控机制以及它们在槲皮素诱导下所发挥的生物学功能进行了研究。通过外源过表达、RNAi技术初步探究表明,EGR-1与ASPP1基因在转录水平以及蛋白水平上存在明显的相关性。初步实验得到两个基因相关性后即进行调控机制的研究,首先利用双荧光素酶报告系统实验(Luciferase assay),外源过表达、下调内源性EGR-1的表达量可以正向调控ASPP1基因(-1040—+88)启动子区,发现其调控的核心区域为(-284—+88);染色质免疫共沉淀的实验(Ch IP)结果表明转录因子EGR-1确实可以结合在ASPP1(-284—+88)这一区域内,与Luciferase assay结果一致,证明了EGR-1可作为转录因子正向调控ASPP1基因的表达。槲皮素作为天然植物提取物,已被许多文献报道具有促进细胞凋亡的作用,而槲皮素的这一功能的具体机制还尚未明确,在前期的实验结果中发现EGR-1和ASPP1的表达量均可以被槲皮素诱导而上升。这些现象提示,作为肿瘤抑制因子的ASPP1蛋白可能被EGR-1调控参与槲皮素促进细胞凋亡这一现象。经过后期ChIP实验表明,受到槲皮素处理后的HCT116细胞中,EGR-1与ASPP1启动子区的结合能力增强,表明EGR-1激活了ASPP1基因的表达。通过RNAi下调了EGR-1和ASPP1蛋白后,发现槲皮素降低细胞活性的能力明显降低。经过流式细胞仪检测验证,这种细胞活力的上升主要是由于细胞凋亡水平被抑制而产生的。本文发现了转录因子EGR-1可以通过结合ASPP1启动子来调控其表达,这为ASPP1的表达调控机制提供了新的解释,也为研究槲皮素引起细胞凋亡的发生机制提供了新的思路和方向。总之,这些发现为了更好的解肿瘤细胞的发生和发展机制,为肿瘤的防治提供了新的靶点和理论基础。
[Abstract]:ASPP1 is a newly discovered suppressor gene of ASPPs, and its expression is down-regulated in many kinds of cancer tissues. Therefore, it is important to reveal the upstream regulation mechanism of ASPP1 in order to explain the decrease of ASPPs expression. At present, the regulation of epigenetic level has mainly been found, and the transcription factors regulating ASPP1 gene at the transcriptional level have only been reported as members of the E2F family. The regulation mechanism of EGR-1 on ASPP1 and its biological function induced by quercetin were studied. A preliminary study of exogenous overexpression RNAi showed that EGR-1 was significantly correlated with ASPP1 gene at the transcriptional and protein levels. The regulatory mechanism of the two genes was studied immediately after the preliminary experiment. Firstly, by using the double luciferase report system (Luciferase assay), down-regulating the expression of endogenous EGR-1 could positively regulate the promoter region of ASPP1 gene (-1040-88). It was found that the core region regulated by EGR-1 was (-284-88), and the results of chromatin immunoprecipitation (Ch IP) showed that EGR-1 could indeed bind to the region of ASPP1 (-284-88), which was consistent with the result of Luciferase assay. It was proved that EGR-1 could be used as a transcription factor to regulate the expression of ASPP1 gene. Quercetin, as a natural plant extract, has been reported in many literatures to promote apoptosis, but the specific mechanism of this function of quercetin has not been clarified. It was found that the expression of EGR-1 and ASPP1 could be increased by quercetin. These results suggest that ASPP1 protein, as a tumor suppressor, may be regulated by EGR-1 and involved in quercetin induced apoptosis. The binding ability of EGR-1 to ASPP1 promoter was enhanced in HCT116 cells treated with quercetin, which indicated that EGR-1 activated the expression of ASPP1 gene. When EGR-1 and ASPP1 proteins were down-regulated by RNAi, the ability of quercetin to decrease cell activity was significantly decreased. Flow cytometry showed that the increase of cell viability was mainly due to the inhibition of apoptosis level. We found that EGR-1 can regulate the expression of EGR-1 by binding to ASPP1 promoter, which provides a new explanation for the expression regulation mechanism of ASPP1, and provides a new idea and direction for studying the mechanism of apoptosis induced by quercetin. In conclusion, these findings provide a new target and theoretical basis for the prevention and treatment of tumor in order to better understand the mechanism of tumor cell formation and development.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R730.5
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本文编号：2075752