本文關(guān)鍵詞：IRES依賴型翻譯增強(qiáng)雙抑癌基因表達(dá)載體對腫瘤的療效　出處：《吉林大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： p14ARF IRES 蛋白質(zhì)翻譯 p53

【摘要】：真核生物細(xì)胞中有兩種翻譯起始模式。絕大多數(shù)真核m RNA通過帽依賴型模式啟動(dòng)相應(yīng)蛋白質(zhì)的合成。在這種機(jī)制中,帽結(jié)構(gòu)與真核翻譯起始因子4E(e IF4E)相互作用對于募集其他翻譯起始因子(e IFs)結(jié)合與m RNA的5'端起著關(guān)鍵作用。以內(nèi)部核糖體進(jìn)入位點(diǎn)(IRES)起始的蛋白質(zhì)翻譯是另一種蛋白質(zhì)翻譯模式,核糖體通過IRES的二級(jí)結(jié)構(gòu)直接被募集并結(jié)合m RNA,而不需要依賴5'-末端帽結(jié)構(gòu)。IRES通常不于e IFs結(jié)合,但能與一類被稱為IRES反式作用因子(ITAFs)的蛋白結(jié)合。IRES依賴型翻譯起始最早在脊髓灰質(zhì)炎病毒RNA和腦心肌炎病毒(EMCV)RNA中被發(fā)現(xiàn),現(xiàn)在認(rèn)為某些細(xì)胞RNA也通過這種機(jī)制進(jìn)行翻譯,特別是在缺氧或細(xì)胞應(yīng)激引發(fā)的異常狀態(tài)下。IRES已經(jīng)引起基因治療領(lǐng)域的興趣,因?yàn)橥ㄟ^它能夠設(shè)計(jì)在一個(gè)啟動(dòng)子下表達(dá)兩個(gè)基因的雙順反子基因轉(zhuǎn)移載體。與細(xì)胞內(nèi)大多數(shù)需要5’端帽結(jié)構(gòu)m RNA的翻譯不同,IRES介導(dǎo)的翻譯不需要帽結(jié)構(gòu)參與。腫瘤抑制因子p53控制細(xì)胞周期停滯和凋亡通路,是腫瘤抑制的中心。該通路是通過p53蛋白穩(wěn)定和轉(zhuǎn)錄激活功能活化而引發(fā)的,以應(yīng)答在正常細(xì)胞異常但在腫瘤細(xì)胞中常見的現(xiàn)象,如癌基因活化和DNA損傷。p53基因在超過50%的腫瘤中發(fā)生缺失或突變,導(dǎo)致p53通路被破壞,因此p53基因的缺失或突變可能是腫瘤發(fā)展中必不可少的一步。所以,p53通路引起了人們的重視,不僅因?yàn)槠鋵δ[瘤細(xì)胞的重要作用,還因其具有腫瘤治療的潛力。但由于p53受激活信號(hào)(如DNA損傷)和調(diào)節(jié)蛋白(如p14ARF和mdm2)等多種因素的調(diào)控,這也解釋了為何在腫瘤細(xì)胞中單獨(dú)過表達(dá)外源性p53并不能總是有效地導(dǎo)致細(xì)胞周期停滯或細(xì)胞凋亡。p14ARF是新近發(fā)現(xiàn)的抑癌基因,在多種腫瘤中存在缺失突變。而且p14ARF能夠與多種蛋白質(zhì)相互作用發(fā)揮抑癌作用。其中一個(gè)被廣泛認(rèn)可的功能是通過活化p53,從而抑制細(xì)胞因癌基因活化導(dǎo)致的異常生長。p14ARF能夠通過抵消mdm2的抑制作用使p53穩(wěn)定和活化。另外,p14ARF還具有不依賴p53途徑的抑制細(xì)胞生長的能力。因此p14ARF也是非常具有潛力的基因治療靶點(diǎn)。我們構(gòu)建Adp14和Adp14/p53重組腺病毒,對比Adp14與Adp53聯(lián)合應(yīng)用和Adp14/p53單獨(dú)應(yīng)用對腫瘤細(xì)胞的抑制作用,找出更有效的處理方法。然后我們對比p14ARF對帽依賴型翻譯和IRES依賴性翻譯的影響。方法:(1)通過Ad Easy TM腺病毒載體系統(tǒng)包裝重組腺病毒Adp14和Adp14/p53;(2)MTT法檢測人結(jié)腸癌細(xì)胞DLD-1生存率;(3)實(shí)時(shí)熒光定量PCR檢測p14ARF m RNA和p53 m RNA表達(dá);(3)Western blot檢測p14、p53、mdm2表達(dá);(4)多聚核糖體分析檢測p53翻譯速率;(5)si RNA沉默DLD-1細(xì)胞內(nèi)mdm2;(6)構(gòu)建攜帶GFP和IRES-GFP的逆轉(zhuǎn)錄病毒,感染人食管癌細(xì)胞OE33,建立穩(wěn)轉(zhuǎn)細(xì)胞系OE33-GFP和OE33-IRES-GFP;(7)[35S]甲硫氨酸標(biāo)記OE33-GFP和OE33-IRES-GFP細(xì)胞內(nèi)GFP,接著免疫共沉淀獲得GFP蛋白進(jìn)行SDS-PAGE和熒光照相。結(jié)果:(1)與Adp14和Adp53單處理組相比,Adp14與Adp53聯(lián)合組DLD-1細(xì)胞能生存率降低。但Adp14與Adp53聯(lián)合組的p53蛋白質(zhì)含量很低;(2)通過多聚核糖體分析發(fā)現(xiàn)Adp14處理的DLD-1細(xì)胞p53的翻譯速率降低,說明過表達(dá)p14ARF能抑制p53翻譯。Adp14/p53處理的DLD-1細(xì)胞p53又恢復(fù)高速翻譯狀態(tài);(3)通過干擾DLD-1細(xì)胞的mdm2基因表達(dá),顯示mdm2的缺失不能使p53翻譯速率下降,排除了mdm2誘導(dǎo)Adp14/p53組DLD-1細(xì)胞內(nèi)p53翻譯速率提高的可能;(4)通過對比Adp14與Adp53聯(lián)合組、Adp14/p53組發(fā)現(xiàn),p53通過不同方式進(jìn)行翻譯。Adp14與Adp53聯(lián)合組通過帽依賴型方式翻譯,Adp14/p53組通過IRES依賴型方式翻譯。DN-MEFs細(xì)胞分別被Adp53、Adp14/p53、Adp14與Adp53聯(lián)合處理,多聚核糖體分析發(fā)現(xiàn)Adp14與Adp53聯(lián)合組p53翻譯速率低,而Adp14/p53組翻譯速率高。過表達(dá)p14ARF時(shí),通過帽依賴型翻譯的p53受到抑制,而通過IRES依賴型翻譯的p53不受影響;(5)構(gòu)建OE33-GFP和OE33-IRES-GFP穩(wěn)轉(zhuǎn)細(xì)胞系,通過[35S]甲硫氨酸標(biāo)記技術(shù)檢測GFP蛋白合成速率。結(jié)果顯示通過帽依賴型翻譯的GFP蛋白合成受到抑制,與對照組比下降了90%左右,而通過IRES依賴型翻譯的GFP蛋白合成僅受到微弱的影響。說明IRES調(diào)節(jié)的翻譯能逃脫p14ARF的翻譯抑制,并且這是涉及p14ARF的基因轉(zhuǎn)移的普遍現(xiàn)象。結(jié)論:(1)Adp14聯(lián)合Adp53同Adp14/p53一樣能夠抑制腫瘤生長,但Adp14/p53的抑制作用更顯著。(2)p14ARF能夠影響帽依賴型機(jī)制進(jìn)行的蛋白質(zhì)翻譯速率,抑制蛋白質(zhì)合成;而IRES依賴型蛋白質(zhì)翻譯能克服p14ARF的翻譯抑制作用。綜上所述,p14ARF不僅是腫瘤抑制蛋白,還具有抑制帽依賴型翻譯起始的功能。這表明在高水平p14ARF存在時(shí),將基因置于IRES下游能夠保持基因產(chǎn)物的高效翻譯,這一發(fā)現(xiàn)可能對腫瘤基因治療策略有重大意義。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R450;R730.5

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