本文選題：槲皮素 + EGR1��； 參考：《哈爾濱工業(yè)大學》2016年碩士論文

【摘要】：ASPP1是新發(fā)現(xiàn)的蛋白家族ASPPs中的一個抑癌基因,其表達量在許多種類的癌癥組織中下調(diào),因此揭示其上游調(diào)控機制對解釋其表達量下降具有重要的意義。目前已發(fā)現(xiàn)的主要是表觀遺傳水平的調(diào)控,而在轉(zhuǎn)錄水平上調(diào)控ASPP1基因的轉(zhuǎn)錄因子現(xiàn)已有報道的僅有E2F家族的成員。本文就EGR-1對ASPP1的調(diào)控機制以及它們在槲皮素誘導下所發(fā)揮的生物學功能進行了研究。通過外源過表達、RNAi技術初步探究表明,EGR-1與ASPP1基因在轉(zhuǎn)錄水平以及蛋白水平上存在明顯的相關性。初步實驗得到兩個基因相關性后即進行調(diào)控機制的研究,首先利用雙熒光素酶報告系統(tǒng)實驗(Luciferase assay),外源過表達、下調(diào)內(nèi)源性EGR-1的表達量可以正向調(diào)控ASPP1基因(-1040—+88)啟動子區(qū),發(fā)現(xiàn)其調(diào)控的核心區(qū)域為(-284—+88);染色質(zhì)免疫共沉淀的實驗(Ch IP)結果表明轉(zhuǎn)錄因子EGR-1確實可以結合在ASPP1(-284—+88)這一區(qū)域內(nèi),與Luciferase assay結果一致,證明了EGR-1可作為轉(zhuǎn)錄因子正向調(diào)控ASPP1基因的表達。槲皮素作為天然植物提取物,已被許多文獻報道具有促進細胞凋亡的作用,而槲皮素的這一功能的具體機制還尚未明確,在前期的實驗結果中發(fā)現(xiàn)EGR-1和ASPP1的表達量均可以被槲皮素誘導而上升。這些現(xiàn)象提示,作為腫瘤抑制因子的ASPP1蛋白可能被EGR-1調(diào)控參與槲皮素促進細胞凋亡這一現(xiàn)象。經(jīng)過后期ChIP實驗表明,受到槲皮素處理后的HCT116細胞中,EGR-1與ASPP1啟動子區(qū)的結合能力增強,表明EGR-1激活了ASPP1基因的表達。通過RNAi下調(diào)了EGR-1和ASPP1蛋白后,發(fā)現(xiàn)槲皮素降低細胞活性的能力明顯降低。經(jīng)過流式細胞儀檢測驗證,這種細胞活力的上升主要是由于細胞凋亡水平被抑制而產(chǎn)生的。本文發(fā)現(xiàn)了轉(zhuǎn)錄因子EGR-1可以通過結合ASPP1啟動子來調(diào)控其表達,這為ASPP1的表達調(diào)控機制提供了新的解釋,也為研究槲皮素引起細胞凋亡的發(fā)生機制提供了新的思路和方向。總之,這些發(fā)現(xiàn)為了更好的解腫瘤細胞的發(fā)生和發(fā)展機制,為腫瘤的防治提供了新的靶點和理論基礎。
[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.
【學位授予單位】：哈爾濱工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R730.5
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本文編號：2075752