本文選題：EIF2AK3　切入點(diǎn)：TMEM216　出處：《山東大學(xué)》2017年碩士論文

【摘要】：肺癌是世界上死亡率最高的癌癥,目前治療肺癌的常用方法仍是化療,但是化療副作用大,因此尋找更具特異性的腫瘤治療靶點(diǎn)顯得尤為重要。近年來(lái),化療藥物通過(guò)引起內(nèi)質(zhì)網(wǎng)應(yīng)激促進(jìn)腫瘤細(xì)胞凋亡的研究已有大量報(bào)道。因此,研究?jī)?nèi)質(zhì)網(wǎng)應(yīng)激的調(diào)控機(jī)制對(duì)于研究腫瘤細(xì)胞凋亡,尋找新的腫瘤治療靶點(diǎn)具有重要的理論意義。EIF2AK3作為重要的未折疊蛋白反應(yīng)的感受器蛋白,目前關(guān)于它的調(diào)控機(jī)制研究主要有蛋白活性調(diào)控和mRNA穩(wěn)定性調(diào)控兩方面,而關(guān)于其降解機(jī)制的研究尚未見(jiàn)報(bào)道。TMEM216對(duì)維持細(xì)胞正常生理狀態(tài)起著非常重要的作用。我們前期數(shù)據(jù)顯示,內(nèi)質(zhì)網(wǎng)應(yīng)激時(shí)TMEM216蛋白量上調(diào);另外有文獻(xiàn)指出TMEM216與EIF2AK3可能存在相互作用。因此,本課題希望以TMEM216為突破口,研究EIF2AK3的調(diào)控機(jī)制,并試圖闡述TMEM216調(diào)控腫瘤細(xì)胞凋亡的分子機(jī)制并尋找新的腫瘤治療靶點(diǎn)。首先,我們檢測(cè)了不同NSCLC細(xì)胞系中TMEM216和EIF2AK3的蛋白水平,發(fā)現(xiàn)二者的蛋白水平可能有關(guān);并且敲低TMEM216后,免疫印跡實(shí)驗(yàn)結(jié)果顯示EIF2AK3及其下游相關(guān)蛋白下調(diào),而對(duì)ERN1的蛋白水平?jīng)]有影響;而過(guò)表達(dá)TMEM216會(huì)上調(diào)EIF2AK3及下游相關(guān)蛋白的水平。這些實(shí)驗(yàn)結(jié)果表明,TMEM216上調(diào)EIF2AK3的蛋白水平。其次,我們運(yùn)用RNA干擾技術(shù)敲低TMEM216的表達(dá),蛋白質(zhì)免疫印跡實(shí)驗(yàn)和流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示細(xì)胞凋亡水平下調(diào);相反過(guò)表達(dá)TMEM216后凋亡水平上調(diào)。這些實(shí)驗(yàn)結(jié)果表明,TMEM216促進(jìn)NSCLC細(xì)胞凋亡。另外我們敲低EIF2AK3后過(guò)表達(dá)TMEM216,進(jìn)行免疫印跡實(shí)驗(yàn),發(fā)現(xiàn)敲低EIF24K3抑制了 TMEM216的促凋亡效應(yīng)。據(jù)此我們得出結(jié)論:TMEM216通過(guò)上調(diào)EIF2AK3促進(jìn)細(xì)胞凋亡。再次,我們通過(guò)免疫共沉淀實(shí)驗(yàn)證實(shí)了 TMEM216與EIF2AK3的確存在相互作用,并調(diào)控EIF2AK3經(jīng)泛素-蛋白酶體途徑的降解。接下來(lái),我們通過(guò)RNA干擾、免疫印跡以及免疫共沉淀實(shí)驗(yàn)篩選出EIF2AK3 一個(gè)可能的E3泛素連接酶:SYVN1;免疫共沉淀及泛素化分析實(shí)驗(yàn)結(jié)果表明TMEM216抑制EIF2AK3與SYVN1結(jié)合,并下調(diào)EIF2AK3的多聚泛素化水平。最后我們初步驗(yàn)證了內(nèi)質(zhì)網(wǎng)應(yīng)激可能會(huì)通過(guò)上調(diào)DDIT3而對(duì)TMEM216進(jìn)行正反饋調(diào)控。綜上所述,我們以NSCLC細(xì)胞為模型,明確了 TMEM216上調(diào)EIF2AK3的蛋白水平,并促進(jìn)細(xì)胞凋亡;首次證明了 EIF2AK3經(jīng)泛素-蛋白酶體途徑降解,并且證明了 SYVN1可能是EIF2AK3的一個(gè)E3連接酶;探究了 TMEM216對(duì)EIF2AK3降解的調(diào)控機(jī)制,即TMEM216抑制EIF2AK3與SYVN1相結(jié)合進(jìn)而下調(diào)其多聚泛素化水平;最后我們驗(yàn)證了內(nèi)質(zhì)網(wǎng)應(yīng)激可能會(huì)通過(guò)上調(diào)DDIT3對(duì)TMEM216進(jìn)行正反饋調(diào)控。
[Abstract]:Lung cancer is the highest mortality cancer in the world. At present, chemotherapy is still commonly used to treat lung cancer, but the side effects of chemotherapy are large, so it is particularly important to find a more specific target for cancer treatment in recent years. It has been reported that chemotherapeutic drugs promote apoptosis of tumor cells by inducing endoplasmic reticulum stress. Therefore, it is important to study the regulation mechanism of endoplasmic reticulum stress on tumor cell apoptosis. Finding a new target for tumor therapy has important theoretical significance. EIF2AK3 is an important receptor protein for unfolded protein reaction. At present, the regulation mechanism of EIF2AK3 is mainly involved in the regulation of protein activity and the regulation of mRNA stability. However, the studies on the degradation mechanism of TMEM216 have not been reported. TMEM216 plays a very important role in maintaining the normal physiological state of cells. Our previous data showed that the amount of TMEM216 protein was up-regulated during endoplasmic reticulum stress. In addition, some literatures indicate that there may be interaction between TMEM216 and EIF2AK3. Therefore, this paper hopes to use TMEM216 as a breakthrough point to study the regulatory mechanism of EIF2AK3, and try to elucidate the molecular mechanism of TMEM216 regulating apoptosis of tumor cells and seek a new target for tumor therapy. We detected the protein levels of TMEM216 and EIF2AK3 in different NSCLC cell lines, and found that the protein levels of TMEM216 and EIF2AK3 might be related, and after knocking down TMEM216, the results of Western blot showed that EIF2AK3 and its downstream related proteins were down-regulated, but had no effect on the protein level of ERN1. Overexpression of TMEM216 upregulated the level of EIF2AK3 and downstream related proteins. These results showed that TMEM216 up-regulated the protein level of EIF2AK3. Secondly, we used RNA interference technique to knock down the expression of TMEM216. The results of Western blot and flow cytometry showed that the level of apoptosis was down-regulated. These results suggest that TMEM216 promotes apoptosis in NSCLC cells. In addition, we overexpression TMEM216after knocking down EIF2AK3, and carried out immunoblotting assay. We found that knockout of EIF24K3 inhibited the apoptosis-promoting effect of TMEM216. Based on this, we concluded that TMEM216 promoted apoptosis by up-regulating EIF2AK3. Thirdly, we confirmed the interaction between TMEM216 and EIF2AK3 by immunoprecipitation. And regulate the degradation of EIF2AK3 via the ubiquitin proteasome pathway. Next, we interfere with RNA, A possible E3-ubiquitin ligase: SYVN1 was screened by immunoblotting and immunoprecipitation assay, and the results of immunoprecipitation and ubiquitin analysis showed that TMEM216 inhibited the binding of EIF2AK3 to SYVN1. Finally, we preliminarily verified that endoplasmic reticulum stress may regulate TMEM216 positively by up-regulating DDIT3. In conclusion, we use NSCLC cells as a model to clarify the up-regulation of EIF2AK3 protein level by TMEM216. It is the first time that EIF2AK3 is degraded by ubiquitin proteasome pathway, and that SYVN1 may be an E3 ligase of EIF2AK3. The regulation mechanism of TMEM216 on EIF2AK3 degradation is explored. That is, TMEM216 inhibited the combination of EIF2AK3 and SYVN1 and down-regulated the level of polyubiquitin. Finally, we verified that endoplasmic reticulum stress might regulate TMEM216 positively by up-regulating DDIT3.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R734.2

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本文編號(hào)：1685222