發(fā)布時間：2018-03-22 23:36

  本文選題：E2Fs　切入點：E2F2　出處：《吉林大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：胃癌是世界上最常見的惡性腫瘤之一,其發(fā)生、發(fā)展是一個復(fù)雜的、涉及多基因的調(diào)控網(wǎng)絡(luò)失衡導(dǎo)致的細(xì)胞異常代謝的結(jié)果。基因的表達(dá)受轉(zhuǎn)錄水平及翻譯水平的調(diào)控,轉(zhuǎn)錄因子(TF)及micro RNA(mi RNA)在調(diào)控基因的表達(dá)方面扮演了重要的角色。TF和mi RNA之間存在著廣泛的相互作用和合作調(diào)控,共同組成復(fù)雜的調(diào)控網(wǎng)絡(luò),對于我們了解細(xì)胞的生理過程,生物學(xué)功能,疾病的發(fā)生機(jī)制等都起到了重要的作用。在胃癌發(fā)展進(jìn)程中,腫瘤細(xì)胞的惡性增殖是其癌癥發(fā)生的始動因素。轉(zhuǎn)錄因子E2F家族在調(diào)控細(xì)胞周期進(jìn)程方面具有重要作用。E2Fs通過調(diào)控G1/S期DNA合成所需的時相基因控制細(xì)胞周期過程。由于細(xì)胞周期紊亂在癌癥發(fā)生過程中的重要作用使得E2Fs與胃癌的發(fā)生密切相關(guān)。E2Fs相關(guān)的調(diào)控網(wǎng)絡(luò)或許可以幫助我們深入了解胃癌的發(fā)生的機(jī)制及胃癌中E2Fs相關(guān)的臨床病理特征。MiRNA可以通過與靶基因結(jié)合,發(fā)揮基因的轉(zhuǎn)錄調(diào)控功能。越來越多的研究表明mi RNA在腫瘤的始動、進(jìn)展及轉(zhuǎn)移等過程中發(fā)揮了重要的調(diào)節(jié)作用。近期很多研究證實mi R-31與多種腫瘤的進(jìn)展及不良預(yù)后密切相關(guān),但其在胃癌中的生物學(xué)功能卻鮮少報道。生物信息學(xué)分析表明,E2F2是mi R-31的直接靶基因。因此,我們猜測mi R-31也許通過靶向調(diào)節(jié)E2F2的表達(dá)水平來調(diào)控腫瘤的進(jìn)展及預(yù)后。本研究通過基因芯片技術(shù)高通量分析胃癌中差異表達(dá)的基因和mi RNA,結(jié)合生物信息學(xué)軟件預(yù)測與E2Fs調(diào)控相關(guān)的TF、mi RNA和靶基因,構(gòu)建胃癌中E2Fs相關(guān)的TF-mi RNA共調(diào)控網(wǎng)絡(luò),分析調(diào)控網(wǎng)絡(luò)中的關(guān)鍵基因及關(guān)鍵mi RNA的功能。此外,通過檢測mi R-31在胃癌組織及細(xì)胞中的表達(dá)水平,及mi R-31與E2F2的靶向調(diào)控關(guān)系,進(jìn)一步探索mi R-31及E2F2對胃癌細(xì)胞多種生物學(xué)功能的影響,從而明確mi R-31及E2F2在胃癌進(jìn)程及生存率等方面的影響。第一部分胃癌中E2Fs相關(guān)TF-mi RNA共調(diào)控網(wǎng)絡(luò)的構(gòu)建通過分析45對胃癌及癌旁正常組織的差異基因表達(dá)譜及15對胃癌及癌旁正常組織的差異mi RNA表達(dá)譜,我們在胃癌組織中篩選到了887個上調(diào)和93個下調(diào)的差異表達(dá)基因,以及41個下調(diào)和4個上調(diào)的差異表達(dá)mi RNA。使用TRED數(shù)據(jù)庫篩選到了105個胃癌中E2F家族調(diào)控的潛在靶基因�；赥argetscan、mi Randa、mi RDB及mi RWalk數(shù)據(jù)庫的預(yù)測,篩選到胃癌中調(diào)控E2Fs的潛在mi RNA�；谛酒瑪�(shù)據(jù)及數(shù)據(jù)庫的篩選,構(gòu)建胃癌中E2Fs相關(guān)的TF-mi RNA共調(diào)控網(wǎng)絡(luò)并篩選出了18個關(guān)鍵基因及mi RNA。此外,我們發(fā)現(xiàn),過表達(dá)的E2Fs與胃癌的進(jìn)展密切相關(guān)。第二部分mi R-31抑制胃癌發(fā)生、發(fā)展的研究基于胃癌中TF-mi RNA共調(diào)控網(wǎng)絡(luò),我們篩選出了mi R-31極有可能介導(dǎo)了E2F2對胃癌進(jìn)展的調(diào)控作用。通過檢測mi R-31在胃癌組織及細(xì)胞的表達(dá)水平發(fā)現(xiàn)mi R-31在40對胃癌及癌旁正常組織中呈現(xiàn)顯著低表達(dá),且其低表達(dá)水平與胃癌的低分化、淋巴結(jié)轉(zhuǎn)移、T分期及不良預(yù)后呈顯著正相關(guān)。使用mi R-31mimics轉(zhuǎn)染胃癌SGC-7901及MGC-803細(xì)胞系,發(fā)現(xiàn)過表達(dá)的mi R-31可以顯著降低腫瘤細(xì)胞的生存能力、增加細(xì)胞凋亡、使細(xì)胞周期阻滯在G1期、降低腫瘤細(xì)胞的遷移及侵襲能力。構(gòu)建mi R-31過表達(dá)的荷瘤小鼠模型,發(fā)現(xiàn)高表達(dá)的mi R-31可以抑制小鼠體內(nèi)移植瘤的生長。第三部分mi R-31調(diào)控E2F2的表達(dá)抑制胃癌發(fā)生、發(fā)展的機(jī)制研究通過q RT-PCR及Western blot檢測,發(fā)現(xiàn)E2F2在胃癌組織中呈現(xiàn)顯著高表達(dá),其高表達(dá)水平與胃癌的低分化、淋巴結(jié)轉(zhuǎn)移、T分期及不良預(yù)后呈顯著負(fù)相關(guān)。熒光素酶報告實驗顯示,E2F2是mi R-31的直接靶基因。E2F2的表達(dá)水平與mi R-31的表達(dá)水平呈顯著負(fù)相關(guān)。使用si RNA E2F2轉(zhuǎn)染SGC-7901及MGC-803細(xì)胞系敲減E2F2的表達(dá),可以呈現(xiàn)與mi R-31過表達(dá)類似的抗腫瘤結(jié)果。表明mi R-31通過靶向調(diào)控E2F2的表達(dá)抑制胃癌的進(jìn)展。
[Abstract]:Gastric cancer is one of the most common malignant tumors in the world. Its occurrence, development is a complex, abnormal cell metabolism leads to imbalance of gene regulatory network involving the results. The gene expression is regulated by the transcription and translation level, transcription factor (TF) and micro RNA (MI RNA) in the regulation of gene expression the play of the interaction and cooperation between the important role of extensive regulation of.TF and MI RNA, composed of a complex regulatory network, for our understanding of the physiological process, cell biology function, disease mechanism plays an important role in the development of gastric cancer. In the process, the malignant proliferation of tumor cells is the initial factor of cancer. The E2F family of transcription factors plays an important role in.E2Fs by regulating G1/S period required for DNA synthesis phase gene controlling the cell cycle process in the regulation of cell cycle progression by hand. In the disorder of cell cycle in cancer an important role in the process of the occurrence of.E2Fs is closely related to the relevant regulatory network may help us understand the clinical and pathological features of.MiRNA E2Fs and the mechanism of the occurrence of gastric cancer related gastric carcinoma can be combined with the target gene E2Fs and gastric cancer, gene transcription regulation functions play a more and more research. Show mi RNA in tumor initiation, play an important role in the process of development and metastasis. Recently many studies have confirmed that MI R-31 with tumor progression and poor prognosis are closely related, but its biological function in gastric carcinoma is rarely reported. Bioinformatics analysis showed that E2F2 is a direct target gene of MI R-31 therefore, we hypothesized that the progress and prognosis of MI by targeting R-31 may regulate the expression of E2F2 regulated tumor. This study by Qualcomm gene chip technology Analysis of differential gene expression in gastric cancer and MI RNA, combined with bioinformatics software prediction and regulation of E2Fs related TF, MI and RNA target gene, construct the co regulation network of TF-mi in human gastric cancer RNA E2Fs, analysis of key genes in the regulatory network and the key function of RNA MI. In addition, through the detection of MI in R-31 gastric cancer tissues and cells and the expression level of MI R-31 and E2F2 targeting regulatory relations, to further explore the effects of MI R-31 and E2F2 of gastric cancer cell biological function, so as to clear the MI R-31 and E2F2 in gastric cancer progression and survival rate were studied. The first part constructs E2Fs in gastric cancer TF-mi RNA co regulation the network through the analysis of difference between MI spectra and RNA 15 of gastric cancer tissues and normal tissues of 45 differentially expressed genes in gastric carcinoma and adjacent normal tissue expression profile in gastric cancer, we screened 887 up-regulated and 93 down regulated Differential gene expression, and the difference between the 41 down regulated and 4 up-regulated expression of MI RNA. using the TRED database to screen the potential target genes of E2F family of 105 gastric cancer control. Based on Targetscan, MI Randa, MI RDB and MI RWalk database prediction, screening to the regulation of E2Fs in gastric cancer screening potential Mi RNA. chip data and based on the database, the construction of TF-mi in gastric cancer RNA E2Fs co regulatory network and selected 18 key genes MI and RNA. in addition, we found that overexpression of E2Fs is closely related with the progression of gastric cancer. The second part of the MI R-31 inhibition of gastric cancer occurrence, development of gastric cancer in TF-mi RNA based on the co regulation network. We selected mi R-31 might mediate the role of E2F2 in regulation of gastric cancer progression. Through detection of MI R-31 expression level in gastric cancer tissues and cells found in MI R-31 in 40 gastric cancer tissues and normal tissues was observed. The expression was significantly lower, and the low expression level and low differentiation of gastric cancer, lymph node metastasis, was positively related to T stage and poor prognosis. The use of MI R-31mimics and MGC-803 SGC-7901 was transfected into gastric cancer cell lines and found that over expression of MI R-31 can significantly reduce tumor cell viability, increased cell apoptosis, cell cycle arrest in the G1 phase, reduce the invasion and migration of tumor cells. Tumor bearing mice model was constructed over expression of MI R-31, found that the high expression of MI R-31 can inhibit the in vivo tumor growth in mice. The expression of the third part of the MI R-31 regulation of E2F2 inhibits gastric cancer, study on the mechanism of development by Q RT-PCR and Western blot detection. E2F2 was found in gastric cancer tissues showed high expression, its high expression level and low differentiation of gastric cancer, lymph node metastasis, was negatively related to T stage and poor prognosis. The experiment shows that the E2F2 luciferase reporter. A significant negative correlation between expression of direct target gene.E2F2 R-31 and Mi Mi R-31. RNA E2F2 expression using Si transfected SGC-7901 and MGC-803 cells to knockdown E2F2 expression, can show similar antitumor results with MI R-31. Mi R-31 showed that the inhibition of gastric cancer progression to regulate the expression of E2F2 by target.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R735.2
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本文編號：1650928