TMEM216在内质网应激介导的肺癌细胞凋亡中的作用机制研究

发布时间：2018-03-30 08:58

本文选题：EIF2AK3　切入点：TMEM216　出处：《山东大学》2017年硕士论文

【摘要】：肺癌是世界上死亡率最高的癌症,目前治疗肺癌的常用方法仍是化疗,但是化疗副作用大,因此寻找更具特异性的肿瘤治疗靶点显得尤为重要。近年来,化疗药物通过引起内质网应激促进肿瘤细胞凋亡的研究已有大量报道。因此,研究内质网应激的调控机制对于研究肿瘤细胞凋亡,寻找新的肿瘤治疗靶点具有重要的理论意义。EIF2AK3作为重要的未折叠蛋白反应的感受器蛋白,目前关于它的调控机制研究主要有蛋白活性调控和mRNA稳定性调控两方面,而关于其降解机制的研究尚未见报道。TMEM216对维持细胞正常生理状态起着非常重要的作用。我们前期数据显示,内质网应激时TMEM216蛋白量上调;另外有文献指出TMEM216与EIF2AK3可能存在相互作用。因此,本课题希望以TMEM216为突破口,研究EIF2AK3的调控机制,并试图阐述TMEM216调控肿瘤细胞凋亡的分子机制并寻找新的肿瘤治疗靶点。首先,我们检测了不同NSCLC细胞系中TMEM216和EIF2AK3的蛋白水平,发现二者的蛋白水平可能有关;并且敲低TMEM216后,免疫印迹实验结果显示EIF2AK3及其下游相关蛋白下调,而对ERN1的蛋白水平没有影响;而过表达TMEM216会上调EIF2AK3及下游相关蛋白的水平。这些实验结果表明,TMEM216上调EIF2AK3的蛋白水平。其次,我们运用RNA干扰技术敲低TMEM216的表达,蛋白质免疫印迹实验和流式细胞术检测结果显示细胞凋亡水平下调;相反过表达TMEM216后凋亡水平上调。这些实验结果表明,TMEM216促进NSCLC细胞凋亡。另外我们敲低EIF2AK3后过表达TMEM216,进行免疫印迹实验,发现敲低EIF24K3抑制了 TMEM216的促凋亡效应。据此我们得出结论:TMEM216通过上调EIF2AK3促进细胞凋亡。再次,我们通过免疫共沉淀实验证实了 TMEM216与EIF2AK3的确存在相互作用,并调控EIF2AK3经泛素-蛋白酶体途径的降解。接下来,我们通过RNA干扰、免疫印迹以及免疫共沉淀实验筛选出EIF2AK3 一个可能的E3泛素连接酶:SYVN1;免疫共沉淀及泛素化分析实验结果表明TMEM216抑制EIF2AK3与SYVN1结合,并下调EIF2AK3的多聚泛素化水平。最后我们初步验证了内质网应激可能会通过上调DDIT3而对TMEM216进行正反馈调控。综上所述,我们以NSCLC细胞为模型,明确了 TMEM216上调EIF2AK3的蛋白水平,并促进细胞凋亡;首次证明了 EIF2AK3经泛素-蛋白酶体途径降解,并且证明了 SYVN1可能是EIF2AK3的一个E3连接酶;探究了 TMEM216对EIF2AK3降解的调控机制,即TMEM216抑制EIF2AK3与SYVN1相结合进而下调其多聚泛素化水平;最后我们验证了内质网应激可能会通过上调DDIT3对TMEM216进行正反馈调控。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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