IRES依赖型翻译增强双抑癌基因表达载体对肿瘤的疗效

发布时间：2017-12-31 01:07

  本文关键词：IRES依赖型翻译增强双抑癌基因表达载体对肿瘤的疗效　出处：《吉林大学》2017年博士论文　论文类型：学位论文

  更多相关文章： p14ARF IRES 蛋白质翻译 p53

【摘要】：真核生物细胞中有两种翻译起始模式。绝大多数真核m RNA通过帽依赖型模式启动相应蛋白质的合成。在这种机制中,帽结构与真核翻译起始因子4E(e IF4E)相互作用对于募集其他翻译起始因子(e IFs)结合与m RNA的5'端起着关键作用。以内部核糖体进入位点(IRES)起始的蛋白质翻译是另一种蛋白质翻译模式,核糖体通过IRES的二级结构直接被募集并结合m RNA,而不需要依赖5'-末端帽结构。IRES通常不于e IFs结合,但能与一类被称为IRES反式作用因子(ITAFs)的蛋白结合。IRES依赖型翻译起始最早在脊髓灰质炎病毒RNA和脑心肌炎病毒(EMCV)RNA中被发现,现在认为某些细胞RNA也通过这种机制进行翻译,特别是在缺氧或细胞应激引发的异常状态下。IRES已经引起基因治疗领域的兴趣,因为通过它能够设计在一个启动子下表达两个基因的双顺反子基因转移载体。与细胞内大多数需要5’端帽结构m RNA的翻译不同,IRES介导的翻译不需要帽结构参与。肿瘤抑制因子p53控制细胞周期停滞和凋亡通路,是肿瘤抑制的中心。该通路是通过p53蛋白稳定和转录激活功能活化而引发的,以应答在正常细胞异常但在肿瘤细胞中常见的现象,如癌基因活化和DNA损伤。p53基因在超过50%的肿瘤中发生缺失或突变,导致p53通路被破坏,因此p53基因的缺失或突变可能是肿瘤发展中必不可少的一步。所以,p53通路引起了人们的重视,不仅因为其对肿瘤细胞的重要作用,还因其具有肿瘤治疗的潜力。但由于p53受激活信号(如DNA损伤)和调节蛋白(如p14ARF和mdm2)等多种因素的调控,这也解释了为何在肿瘤细胞中单独过表达外源性p53并不能总是有效地导致细胞周期停滞或细胞凋亡。p14ARF是新近发现的抑癌基因,在多种肿瘤中存在缺失突变。而且p14ARF能够与多种蛋白质相互作用发挥抑癌作用。其中一个被广泛认可的功能是通过活化p53,从而抑制细胞因癌基因活化导致的异常生长。p14ARF能够通过抵消mdm2的抑制作用使p53稳定和活化。另外,p14ARF还具有不依赖p53途径的抑制细胞生长的能力。因此p14ARF也是非常具有潜力的基因治疗靶点。我们构建Adp14和Adp14/p53重组腺病毒,对比Adp14与Adp53联合应用和Adp14/p53单独应用对肿瘤细胞的抑制作用,找出更有效的处理方法。然后我们对比p14ARF对帽依赖型翻译和IRES依赖性翻译的影响。方法:(1)通过Ad Easy TM腺病毒载体系统包装重组腺病毒Adp14和Adp14/p53;(2)MTT法检测人结肠癌细胞DLD-1生存率;(3)实时荧光定量PCR检测p14ARF m RNA和p53 m RNA表达;(3)Western blot检测p14、p53、mdm2表达;(4)多聚核糖体分析检测p53翻译速率;(5)si RNA沉默DLD-1细胞内mdm2;(6)构建携带GFP和IRES-GFP的逆转录病毒,感染人食管癌细胞OE33,建立稳转细胞系OE33-GFP和OE33-IRES-GFP;(7)[35S]甲硫氨酸标记OE33-GFP和OE33-IRES-GFP细胞内GFP,接着免疫共沉淀获得GFP蛋白进行SDS-PAGE和荧光照相。结果:(1)与Adp14和Adp53单处理组相比,Adp14与Adp53联合组DLD-1细胞能生存率降低。但Adp14与Adp53联合组的p53蛋白质含量很低;(2)通过多聚核糖体分析发现Adp14处理的DLD-1细胞p53的翻译速率降低,说明过表达p14ARF能抑制p53翻译。Adp14/p53处理的DLD-1细胞p53又恢复高速翻译状态;(3)通过干扰DLD-1细胞的mdm2基因表达,显示mdm2的缺失不能使p53翻译速率下降,排除了mdm2诱导Adp14/p53组DLD-1细胞内p53翻译速率提高的可能;(4)通过对比Adp14与Adp53联合组、Adp14/p53组发现,p53通过不同方式进行翻译。Adp14与Adp53联合组通过帽依赖型方式翻译,Adp14/p53组通过IRES依赖型方式翻译。DN-MEFs细胞分别被Adp53、Adp14/p53、Adp14与Adp53联合处理,多聚核糖体分析发现Adp14与Adp53联合组p53翻译速率低,而Adp14/p53组翻译速率高。过表达p14ARF时,通过帽依赖型翻译的p53受到抑制,而通过IRES依赖型翻译的p53不受影响;(5)构建OE33-GFP和OE33-IRES-GFP稳转细胞系,通过[35S]甲硫氨酸标记技术检测GFP蛋白合成速率。结果显示通过帽依赖型翻译的GFP蛋白合成受到抑制,与对照组比下降了90%左右,而通过IRES依赖型翻译的GFP蛋白合成仅受到微弱的影响。说明IRES调节的翻译能逃脱p14ARF的翻译抑制,并且这是涉及p14ARF的基因转移的普遍现象。结论:(1)Adp14联合Adp53同Adp14/p53一样能够抑制肿瘤生长,但Adp14/p53的抑制作用更显著。(2)p14ARF能够影响帽依赖型机制进行的蛋白质翻译速率,抑制蛋白质合成;而IRES依赖型蛋白质翻译能克服p14ARF的翻译抑制作用。综上所述,p14ARF不仅是肿瘤抑制蛋白,还具有抑制帽依赖型翻译起始的功能。这表明在高水平p14ARF存在时,将基因置于IRES下游能够保持基因产物的高效翻译,这一发现可能对肿瘤基因治疗策略有重大意义。
[Abstract]:There are two kinds of translation initiation mode in eukaryotic cells. Most eukaryotic m RNA through the cap dependent mode synthesis of corresponding proteins. In this mechanism, and the cap structure of eukaryotic translation initiation factor 4E (E IF4E) interaction for raising other translation initiation factor (E IFs) combined with m RNA 5'plays a key role. The internal ribosome entry site (IRES) the initiation of protein translation is another protein translation, the ribosome through two level structure of IRES and m RNA are recruited directly, without the need to rely on the 5'- terminal of the.IRES cap structure is usually not in the E binding to IFs, but with a kind of called the IRES transactivator (ITAFs) dependent translation initiation at the earliest poliovirus RNA and encephalomyocarditis virus binding protein.IRES (EMCV) was found in RNA, now that some cells RNA through this mechanism in translation, especially Is the abnormal state caused by hypoxia or cellular stress under.IRES has caused the field of gene therapy because of interest, it can design the bicistronic gene transfer vector two gene expression in a promoter. Unlike most cells need 5 'end cap structure m RNA IRES mediated translation the translation does not need the cap structure involved. The tumor suppressor p53 cell cycle arrest and apoptosis pathway, tumor suppressor center. The pathway is through p53 protein stability and transcriptional activation in response to activation caused by abnormalities in normal cells but common in tumor cells of the phenomenon, such as oncogene activation and DNA damage more than 50% of the.P53 gene in tumor mutations leading to p53 pathway is damaged, so the absence or mutation of p53 gene may be an essential step in the development of tumor. Therefore, the p53 pathway has aroused people Attention, not only because of its important role in tumor cells, but also because of its tumor therapeutic potential. But because of p53 by activating signals (such as DNA damage) and regulatory proteins (p14ARF and MDM2) Regulation and other factors, this also explains why in tumor cells expressing exogenous p53 alone it is not always effectively lead to cell cycle arrest or apoptosis of.P14ARF tumor suppressor gene discovered recently, mutations in a variety of tumors. And the p14ARF can interact with a variety of proteins play a tumor suppressor role. One widely recognized function is through the activation of p53, thereby inhibiting the abnormal growth of.P14ARF cells for cancer gene through activation leads to inhibition of MDM2 to offset p53 stabilization and activation. In addition, p14ARF also has the ability to inhibit cell growth is not dependent on p53 pathway. Therefore p14ARF is also very has the potential Gene therapy targeting force. We construct Adp14 and Adp14/p53 recombinant adenovirus, compared with Adp14 combined with Adp53 and Adp14/p53 alone inhibitory effect on tumor cells, to find more effective processing method. Then we compared the p14ARF of cap dependent translation and IRES dependent effect of translation. Methods: (1) through Adp14 Adp14/p53 and Ad Easy TM adenovirus vector system packaging recombinant adenovirus; (2) MTT assay of human colon cancer cells DLD-1 survival rate; (3) p14ARF m real time fluorescence quantitative PCR detection of RNA and p53 m RNA expression; (3) Western blot detection of p14, p53, MDM2 expression; (4) analysis p53 translation rate of polysomes; (5) Si RNA in MDM2 silenced DLD-1 cells; (6) carrying GFP and IRES-GFP retrovirus infection of human esophageal cancer cells OE33, to establish stable cell lines OE33-GFP and OE33-IRES-GFP; (7) [35S] markers OE33-GFP and OE33-IRES- methionine GFP GFP in the cells, then co immunoprecipitation of SDS-PAGE and fluorescence photographic GFP protein. Results: (1) compared with Adp14 and Adp53 treatment group, Adp14 group and Adp53 combined with DLD-1 cells can reduce the survival rate of p53. But the protein content of Adp14 and Adp53 combined group is very low; (2) through the poly the analysis found that DLD-1 cells p53 Adp14 ribosome translation processing rate decreased, indicated that the over expression of p14ARF can inhibit DLD-1 cell p53 p53.Adp14/p53 treatment and recovery of high speed translation translation; (3) the MDM2 gene interference cell DLD-1 expression shows that deletion of MDM2 can make the p53 translation rate, excluding MDM2 DLD-1 cells induced by Adp14/p53 in the group p53 translation rate increased; (4) compared with the Adp14 and Adp53 group, Adp14/p53 group, p53 group and Adp53 combined with.Adp14 in different ways through the cap dependent translation mode, Adp14/p53 group IRES dependent translation of.DN-MEFs cells were Adp53, Adp14/p53, Adp14 and Adp53 combined treatment, polysome analysis showed that Adp14 and Adp53 combined with p53 group translation rate is low, and the Adp14/p53 group. The high translation rate of over expression of p14ARF, through the cap dependent translation of p53 suppressed, and by IRES dependent translation the p53 is not affected; (5) to construct OE33-GFP and OE33-IRES-GFP stable cell lines by [35S] methionine labeled technique was used to detect GFP protein synthesis rate. The results showed that GFP protein synthesis through the cap dependent translation is inhibited, and the control group decreased by about 90%, the synthesis of GFP protein by IRES dependent translation only weak influence. The results showed that IRES regulated translation can escape p14ARF translation inhibition, and this is a common phenomenon involving gene transfer of p14ARF. Conclusion: (1) Adp14 combined with Adp53 can inhibit the same as Adp14/ p53 For tumor growth, but the effect of Adp14/p53 was more significant. (2) p14ARF can influence the mechanism of cap dependent protein translation rate, inhibition of protein synthesis; IRES dependent protein translation can overcome p14ARF translation inhibition. In summary, p14ARF is not only the tumor suppressor protein, but also inhibit cap dependent translation initiation. Function. This shows that in the presence of high levels of p14ARF, efficient translation will be placed in the IRES downstream genes can maintain the gene product, this finding may be of great significance for tumor gene therapy strategy.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R450;R730.5

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