eIF4E磷酸化促翻译及与蛋白酶体共抑制致结肠癌细胞凋亡的机制研究

发布时间：2018-08-02 17:30

【摘要】：背景结肠癌是恶性肿瘤中发病率及病死率最高的疾病之一,然而近年来结肠癌患者的存活率并无太大改善。结肠癌患者往往对传统治疗产生耐药性,因此新的治疗策略迫在眉睫。蛋白质翻译起始为一类进化保守的细胞生物过程,受营养/生长因子和细胞能量水平影响而调控细胞生长和新陈代谢。肿瘤细胞快速生长增殖伴随PI3K/Akt/m TOR和Ras/Raf/MEK/ERK等信号通路的激活,蛋白质翻译起始处于这些信号通路的下游枢纽位置。其中,e IF4E是翻译起始复合物中受到多层面精确调控的一个蛋白,其mRNA和蛋白质表达水平在多种肿瘤细胞中上调。除了在转录和翻译水平受到调控,e IF4E还在翻译后修饰水平受到更加精细和快速的调节。在某些肿瘤模型中,只有磷酸化修饰的e IF4E才能发挥其最大活性并诱导肿瘤发生发展。在此基础上,我们推测e IF4E S209磷酸化修饰在结直肠癌发生发展中发挥重要的作用,为了证实这一假设,本研究拟构建eIF4E S209A KI结肠癌细胞株,并比较e IF4E KI和WT两种HCT116结肠癌细胞生长和新陈代谢情况;同时使用polysome实验比较两种细胞中分子水平的变化并探讨其调控机制,从而系统性阐述eIF4E S209磷酸化修饰在结肠癌发生发展中的作用和机制,为靶向eIF4E磷酸化修饰的抗癌新药研发奠定理论基础。由于多种癌基因和抑癌基因调控蛋白质翻译起始过程,因此这一生物过程的失调表现在多种癌症和疾病中。目前靶向蛋白质翻译起始的治疗策略已成为一个引人注目的焦点。一些临床前期研究和临床研究表明真核细胞起始因子e IF4A抑制剂Episilvestrol及其类似物Silvestrol均可杀伤多种癌细胞,其缺陷表现为蛋白质翻译起始也参与正常的细胞生物过程,高浓度翻译起始抑制剂可引发严重的副反应。因此联合应用蛋白质翻译起始抑制剂和其他药物成为结肠癌治疗的一个选择。Bortezomib是常用的药物增敏剂,在本研究中我们发现Bortezomib也可协同Episilvestrol诱导更加显著的结肠癌细胞凋亡。目的:本研究旨在探索e IF4E磷酸化修饰在蛋白质翻译中的重要作用并研究靶向蛋白质翻译抑制剂Episilvestrol协同蛋白酶体抑制剂Bortezomib诱导结肠癌细胞凋亡的作用及机制。方法和材料:(1)通过腺病毒系统构建e IF4E磷酸化修饰KI细胞系,并利用帽依赖性报告基因、免疫共沉淀和m7 pull down实验验证eIF4E KI细胞系对mRNA翻译的影响;(2)利用MTT、2D/3D克隆形成实验、Brd U掺入实验、ds RED、GFP-LC3、流式细胞、裸鼠实验等研究eIF4E KI对体内外细胞生长和新陈代谢的影响;(3)利用polysome实验研究e IF4E KI对细胞内mRNA翻译的影响;(4)通过MTS,Crystal violet和克隆形成试验研究不同浓度Episilvestrol对HCT116结肠癌细胞的生长抑制和杀伤作用。(5)通过定量PCR和Western blot实验研究高浓度Episilvestrol对凋亡信号通路和线粒体应激反应的诱导作用。(6)通过MTS,结晶紫染色和克隆形成试验研究低浓度Episilvestrol协同Bortezomib诱导结肠癌细胞生长抑制和凋亡的作用。(4)使用Z-VAD抑制剂证实Episilvestrol协同Bortezomib诱导结肠癌细胞凋亡的作用。(5)通过定量PCR和Western blot实验研究Episilvestrol协同Bortezomib诱导细胞凋亡/线粒体应激反应和CHOP介导DR5上调的作用。(6)利用FADD敲除细胞研究Episilvestrol协同Bortezomib诱导外源性细胞凋亡。(7)通过MTS,结晶紫染色,克隆形成,定量PCR和Western blot试验研究Episilvestrol协同Bortezomib诱导其他结肠癌细胞凋亡的作用。(8)通过裸鼠移植瘤实验验证Episilvestrol协同Bortezomib诱导结肠癌细胞凋亡的作用。结果:(1)通过腺病毒系统成功构建eIF4E磷酸化修饰KI细胞系;并证实eIF4E KI抑制m RNA翻译;(2)e IF4E KI抑制体内外结肠癌细胞生长和新陈代谢反应;(3)e IF4E与p-eIF4E优先翻译不同群体的m RNA;(4)2.5nM Episilvestrol对HCT116结肠癌细胞无作用,5n M,10n M,15n MEpisilvestrol可引起HCT116结肠癌细胞的生长抑制作用,20n M,25n M Episilvestrol可诱导HCT116结肠癌细胞凋亡。(5)25n M Episilvestrol可抑制HCT116结肠癌细胞克隆形成,并诱导caspase-3/8,DR5,CHOP的表达。(6)2.5nM Episilvestrol联合5nMBortezomib可有效引发HCT116结肠癌细胞凋亡,细胞凋亡抑制剂Z-VAD可抑制这一作用。(7)2.5nM Episilvestrol联合5nMBortezomib在4h即可上调HCT116结肠癌细胞中DR5,CHOP,GADD34的表达,24h作用更加显著。(8)DR5的上调为CHOP介导的,使用CHOP siRNA下调(knockdown)CHOP表达可抑制DR5的上调作用。(9)FADD敲除细胞可完全抑制Episilvestrol联合Bortezomib引发的细胞凋亡作用。(10)2.5nM Episilvestrol联合5n MBortezomib可在其他结肠癌细胞中发挥类似的杀伤作用。(11)在裸鼠移植瘤试验中,单药Episilvestrol,Bortezomib对移植瘤的生长影响甚微,二者联合可有效抑制移植瘤生长。结论:这些结果表明肿瘤细胞利用超过正常生理水平的eIF4E磷酸化来达到在营养缺失等应激条件下的生存。联合使用蛋白质翻译抑制剂Episilvestrol和蛋白酶体抑制剂Bortezomib可作为治疗结肠癌患者的一种策略。
[Abstract]:Background colon cancer is one of the highest morbidity and fatality rates in malignant tumors. However, the survival rate of colon cancer patients has not been greatly improved in recent years. Patients with colon cancer often have resistance to traditional treatment, so the new treatment strategy is imminent. Cell growth and metabolism are regulated by growth factors and cell energy levels. The rapid growth and proliferation of tumor cells are associated with the activation of signal pathways such as PI3K/Akt/m TOR and Ras/Raf/MEK/ERK, and protein translation begins at the downstream hub of these signaling pathways. Among them, e IF4E is a multidimensional precision in the starting complex of translation. A protein that regulates the expression level of mRNA and protein in a variety of tumor cells. In addition to the regulation of transcriptional and translation levels, e IF4E is also more finely and quickly regulated after the translation. In some tumor models, only phosphorylated e IF4E can play its maximum activity and induce tumor hair. On this basis, we speculate that e IF4E S209 phosphorylation modification plays an important role in the development of colorectal cancer. In order to confirm this hypothesis, this study intends to construct a eIF4E S209A KI colon cancer cell line and compare the growth and metabolism of two HCT116 colon cancer cells of E IF4E KI and WT; meanwhile, the polysome experiment ratio is also used. The changes in the molecular level of the two cells and its regulatory mechanism are discussed, which systematically expounds the role and mechanism of eIF4E S209 phosphorylation in the development of colon cancer. It lays a theoretical basis for the development of a new anticancer drug targeted by eIF4E phosphorylation. The maladjustment of this biological process is manifested in a variety of cancers and diseases. The therapeutic strategy targeting the initiation of protein translation has become a noticeable focus. Some preclinical and clinical studies have shown that the eukaryotic initiation factor E IF4A inhibitor Episilvestrol and its analogous Silvestrol can kill a variety of cancer cells. The defect shows that the protein translation initiation is also involved in normal cell biological processes, and the high concentration translation initiation inhibitor can cause serious side effects. Therefore, the combination of protein translation initiation inhibitor and other drugs as a choice for colon cancer treatment is a common drug sensitizer. In this study, we found that.Bortezomib is a common drug sensitizer. Bortezomib can also induce more significant apoptosis of colon cancer cells in conjunction with Episilvestrol. Purpose: This study aims to explore the important role of E IF4E phosphorylation in protein translation and to study the role and mechanism of the protein translation inhibitor Episilvestrol synergistically with proteasome inhibitor Bortezomib to induce the apoptosis of colon cancer cells. Methods and materials: (1) construct e IF4E phosphorylated KI cell lines through adenovirus system, and use caps dependent reporter gene, immunoprecipitation and M7 pull down test to verify the effect of eIF4E KI cell line on mRNA translation; (2) MTT, 2D/3D clone formation experiments, Brd experiments, flow cells, nude mice experiments and so on The effect of eIF4E KI on cell growth and metabolism in vitro and in vivo; (3) the effect of E IF4E KI on intracellular mRNA translation was studied by polysome experiment. (4) the inhibitory and killing effects of different concentrations Episilvestrol on the growth of colorectal cancer cells were studied by MTS, Crystal violet and clone formation. (5) The effect of high concentration of Episilvestrol on the apoptosis signaling pathway and mitochondrial stress response was investigated. (6) the effects of low concentration of Episilvestrol on growth inhibition and apoptosis induced by Bortezomib were studied by MTS, crystal violet staining and clone formation. (4) Z-VAD inhibitors were used to confirm that Episilvestrol was induced with Bortezomib. The role of inducing apoptosis of colon cancer cells. (5) the effect of Episilvestrol synergistic Bortezomib induced apoptosis / mitochondrial stress response and CHOP mediated up regulation of DR5 was studied by quantitative PCR and Western blot. (6) Episilvestrol synergistic Bortezomib induced exogenous apoptosis using FADD knockout cells. (7) through MTS, crystal violet staining, Clone formation, quantitative PCR and Western blot test to study the effect of Episilvestrol synergistic Bortezomib on the apoptosis of other colon cancer cells. (8) the effect of Episilvestrol on the apoptosis of colon cancer cells induced by Bortezomib in nude mice was verified by Bortezomib. Results: (1) the KI cell line modified by eIF4E phosphorylation was successfully constructed by adenovirus system. And eIF4E KI inhibits m RNA translation; (2) e IF4E KI inhibits the growth and metabolism of colon cancer cells in vivo and in vivo and in vivo; (3) e IF4E and p-eIF4E give priority to the translation of M RNA in different groups; (4) 25N M Episilvestrol can induce apoptosis in HCT116 colon cancer cells. (5) 25N M Episilvestrol can inhibit the formation of HCT116 colon cancer cells, and induce caspase-3/8, DR5, CHOP expression. (6) the apoptosis of colorectal cancer cells can be induced by the joint 2.5nM. (7) 2.5 NM Episilvestrol combined with 5nMBortezomib in 4H can increase the expression of DR5, CHOP, GADD34 in HCT116 colon cancer cells, and the effect of 24h is more significant. (8) the up regulation of DR5 is mediated by CHOP. Cell apoptosis. (10) 2.5nM Episilvestrol combined with 5N MBortezomib can play a similar killing effect in other colon cancer cells. (11) in nude mice transplantation tumor test, the single drug Episilvestrol and Bortezomib have little influence on the growth of the transplanted tumor. The two combination can effectively inhibit the growth of the transplanted tumor. Conclusion: These results indicate that the tumor cells are beneficial. The use of eIF4E phosphorylation, which is more than normal physiological level, is used to survive under stress conditions such as nutritional deficiency. The combination of protein translation inhibitor Episilvestrol and proteasome inhibitor Bortezomib can be used as a strategy for the treatment of colon cancer patients.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.35

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