MicroRNA-31通过调控E2F2的表达抑制胃癌发生、发展的机制研究

发布时间：2018-03-22 23:36

  本文选题：E2Fs　切入点：E2F2　出处：《吉林大学》2016年博士论文　论文类型：学位论文

【摘要】：胃癌是世界上最常见的恶性肿瘤之一,其发生、发展是一个复杂的、涉及多基因的调控网络失衡导致的细胞异常代谢的结果。基因的表达受转录水平及翻译水平的调控,转录因子(TF)及micro RNA(mi RNA)在调控基因的表达方面扮演了重要的角色。TF和mi RNA之间存在着广泛的相互作用和合作调控,共同组成复杂的调控网络,对于我们了解细胞的生理过程,生物学功能,疾病的发生机制等都起到了重要的作用。在胃癌发展进程中,肿瘤细胞的恶性增殖是其癌症发生的始动因素。转录因子E2F家族在调控细胞周期进程方面具有重要作用。E2Fs通过调控G1/S期DNA合成所需的时相基因控制细胞周期过程。由于细胞周期紊乱在癌症发生过程中的重要作用使得E2Fs与胃癌的发生密切相关。E2Fs相关的调控网络或许可以帮助我们深入了解胃癌的发生的机制及胃癌中E2Fs相关的临床病理特征。MiRNA可以通过与靶基因结合,发挥基因的转录调控功能。越来越多的研究表明mi RNA在肿瘤的始动、进展及转移等过程中发挥了重要的调节作用。近期很多研究证实mi R-31与多种肿瘤的进展及不良预后密切相关,但其在胃癌中的生物学功能却鲜少报道。生物信息学分析表明,E2F2是mi R-31的直接靶基因。因此,我们猜测mi R-31也许通过靶向调节E2F2的表达水平来调控肿瘤的进展及预后。本研究通过基因芯片技术高通量分析胃癌中差异表达的基因和mi RNA,结合生物信息学软件预测与E2Fs调控相关的TF、mi RNA和靶基因,构建胃癌中E2Fs相关的TF-mi RNA共调控网络,分析调控网络中的关键基因及关键mi RNA的功能。此外,通过检测mi R-31在胃癌组织及细胞中的表达水平,及mi R-31与E2F2的靶向调控关系,进一步探索mi R-31及E2F2对胃癌细胞多种生物学功能的影响,从而明确mi R-31及E2F2在胃癌进程及生存率等方面的影响。第一部分胃癌中E2Fs相关TF-mi RNA共调控网络的构建通过分析45对胃癌及癌旁正常组织的差异基因表达谱及15对胃癌及癌旁正常组织的差异mi RNA表达谱,我们在胃癌组织中筛选到了887个上调和93个下调的差异表达基因,以及41个下调和4个上调的差异表达mi RNA。使用TRED数据库筛选到了105个胃癌中E2F家族调控的潜在靶基因。基于Targetscan、mi Randa、mi RDB及mi RWalk数据库的预测,筛选到胃癌中调控E2Fs的潜在mi RNA。基于芯片数据及数据库的筛选,构建胃癌中E2Fs相关的TF-mi RNA共调控网络并筛选出了18个关键基因及mi RNA。此外,我们发现,过表达的E2Fs与胃癌的进展密切相关。第二部分mi R-31抑制胃癌发生、发展的研究基于胃癌中TF-mi RNA共调控网络,我们筛选出了mi R-31极有可能介导了E2F2对胃癌进展的调控作用。通过检测mi R-31在胃癌组织及细胞的表达水平发现mi R-31在40对胃癌及癌旁正常组织中呈现显著低表达,且其低表达水平与胃癌的低分化、淋巴结转移、T分期及不良预后呈显著正相关。使用mi R-31mimics转染胃癌SGC-7901及MGC-803细胞系,发现过表达的mi R-31可以显著降低肿瘤细胞的生存能力、增加细胞凋亡、使细胞周期阻滞在G1期、降低肿瘤细胞的迁移及侵袭能力。构建mi R-31过表达的荷瘤小鼠模型,发现高表达的mi R-31可以抑制小鼠体内移植瘤的生长。第三部分mi R-31调控E2F2的表达抑制胃癌发生、发展的机制研究通过q RT-PCR及Western blot检测,发现E2F2在胃癌组织中呈现显著高表达,其高表达水平与胃癌的低分化、淋巴结转移、T分期及不良预后呈显著负相关。荧光素酶报告实验显示,E2F2是mi R-31的直接靶基因。E2F2的表达水平与mi R-31的表达水平呈显著负相关。使用si RNA E2F2转染SGC-7901及MGC-803细胞系敲减E2F2的表达,可以呈现与mi R-31过表达类似的抗肿瘤结果。表明mi R-31通过靶向调控E2F2的表达抑制胃癌的进展。
[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.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2
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本文编号：1650928