【作者】 马艳红；

【导师】 谭国林；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 叶酸受体a (FOLR1)是细胞摄取叶酸的高亲和性受体,课题组前期研究发现FOLR1的表达与鼻咽癌的发生及紫杉醇耐药表型密切相关,而FOLR1引起的鼻咽癌紫杉醇耐药表型似乎与其具有叶酸转运的功能无关。本文将以鼻咽癌亲本细胞系和紫杉醇耐药细胞系为研究对象,从蛋白质之间的相互作用为切入点,探索FOLR1的作用通路与鼻咽癌紫杉醇耐药之间的相关性。首先以FOLR1多克隆抗体为诱饵通过免疫共沉淀技术筛选其相互作用蛋白,初步确定了在鼻咽癌紫杉醇耐药细胞中FOLR1的相互作用蛋白——角蛋白17(Cytokeratin17,CK17),接着用免疫共沉淀、Western Blot、免疫荧光共定位等技术鉴定相互作用蛋白质,确定在鼻咽癌亲本细胞和紫杉醇耐药细胞中FOLR1与CK17存在功能上的蛋白质间相互作用。通过生物信息学网络分析相互作用蛋白的作用机制,分析FOLR1可能的作用通路,发现二者之间有一个共同的相互作用蛋白——泛素C(ubiquitin C,UBC),二者的相互作用蛋白涉及细胞分化、细胞周期、DNA修饰、信号转导等功能。然后再通过siRNA干扰技术沉默FOLR1或CK17,检测作用通路分子的活化情况,发现沉默FOLR1基因,CK17在转录与蛋白水平表达均下调,而沉默CK17基因,FOLR1的表达无变化,分析FOLR1引起的鼻咽癌紫杉醇耐药表型可能通过FOLR1与CK17间的相互作用这条作用通路,而二者间的相互作用可能通过UBC起作用。我们推断鼻咽癌紫杉醇耐药细胞中FOLR1表达的增高,可能是通过蛋白质的相互作用,诱导和调节与肿瘤细胞生长或凋亡相关的信号分子而发挥作用。图45幅,表21个,参考文献87篇1FOLR1的相互作用蛋白筛选目的：筛选人鼻咽癌紫杉醇耐药细胞中FOLR1的相互作用蛋白。方法：以免疫共沉淀、SDS-PAGE电泳和凝胶染色方法筛选体外培养的正常鼻咽上皮细胞NP69和三株鼻咽癌紫杉醇耐药细胞(CNE-1/Taxol、CNE-2/Taxol、HNE-2/Taxol)中FOLR1的相互作用蛋白,质谱分析方法鉴定相互作用蛋白。结果：CNE-2/taxol细胞蛋白质经过FOLR1的抗原抗体复合物及Protein A琼脂糖珠的共沉淀后,与对照比较,有5条差异条带,分子量分别约为120kD,58kD,50kD,44kD,30kD; CNE-1/taxol10细胞蛋白质经免疫共沉淀后电泳,与对照比较,有3差异条带,分子量分别约为60kD,55kD,50kD; CNE-1/taxol13细胞蛋白质经免疫共沉淀后电泳,与自身对照比较,有2差异条带,分子量分别约为50kD,36kD。结合3次免疫共沉淀实验结果,不同凝胶上都显示出-50kD的差异条带,预示SDS-PAGE凝胶上-50kD的蛋白质可能是与FOLR1相互作用的蛋白质。通过对差异条带进行飞行质谱分析,结果显示该蛋白质为cytokeratin17。结论：人鼻咽癌紫杉醇耐药细胞中FOLR1蛋白可能与CK17蛋白有相互作用。2FOLR1与CK17蛋白相互作用的验证目的：验证筛选出来的FOLR1的相互作用蛋白CK17,确定二者在生理条件下存在相互作用。方法：以免疫共沉淀、Western blot方法验证鼻咽癌亲本细胞(CNE-1)及鼻咽癌紫杉醇耐药细胞(CNE-1/Taxol)中FOLR1与CK17是否存在相互作用；免疫荧光双标法分析FOLR1与CK17的共定位关系；运用Visant软件对生物信息网络数据库进行检索和分析,确定FOLR1和CK17已知的可能相互作用蛋白。结果：1.以FOLR1抗体对鼻咽癌紫杉醇耐药细胞CNE-1/taxol的总蛋白进行免疫共沉淀后,复合物中可以检测到CK17的表达,阴性对照中没有检测到CK17的表达。2.以CK17抗体对鼻咽癌紫杉醇耐药细胞CNE-1/taxol的总蛋白进行免疫共沉淀后,复合物中可以检测到FOLR1的表达,而在对照细胞NP69中没有检测到FOLR1的表达。3.免疫荧光激光共聚焦显微镜观察到FOLR1蛋白在细胞浆及细胞膜中均有表达,CK17蛋白在细胞浆中较强表达,两种蛋白在胞浆中融合。4.生物信息网络Visant软件搜索发现,FOLR1的相互作用蛋白有5个,分别是LYN、UBC、NCAPH2、IRAK3、CUL3、CK17的相互作用蛋白有16个,分别是UBC、YWHAQ、KRT8、KRT72、SNAPAP、 KRT7、EGFR、CCDC85B、KRT6A、APC、GABARAP1、GRB2、 USP1、UCHL1、COPS5、SUM02。2种蛋白质具有共同的相互作用蛋白质,泛素C (Ubiquitin C)结论：1.在鼻咽癌细胞和鼻咽癌紫杉醇耐药细胞中FOLR1与CK17存在物理上的相互作用。2.FOLR1与CK17有一个共同的相互作用蛋白——泛素C。3FOLR1与CK17的功能相互作用目的：通过RNA干扰技术抑制FOLR1或CK17基因表达后,观察信号通路的基因表达变化。方法：采用real time PCR、Western blot方法检测CK17在鼻咽癌亲本细胞(CNE-1)及其紫杉醇耐药细胞(CNE-1/Taxol)中的表达水平；通过RNA干扰技术,沉默CNE-1细胞中CK17的表达,观察FOLR1的变化；沉默CNE-1/Taxol细胞中FOLR1的表达,观察CK17的变化；基因芯片技术分析FOLR1基因沉默后耐药细胞基因组的mRNA表达变化。结果：CK17mRNA和蛋白在鼻咽癌紫杉醇耐药细胞CNE-1/Taxol中表达明显减弱；鼻咽癌细胞CNE-1的CK17基因表达被降低后,FOLR1基因的表达无显著变化；然而,鼻咽癌紫杉醇耐药细胞CNE-1/Taxol的FOLR1基因被降低后,CK17基因的mRNA及蛋白表达水平也显著下调。cDNA Microarray基因芯片结果显示,FOLR1基因沉默后,与FOLR1有相互作用的5个基因表达均下调1.0-1.7倍,CK17表达下调约2.5倍。结论：1. Cytokeratin17在人永生化鼻咽上皮细胞、鼻咽癌亲本细胞、鼻咽癌紫杉醇耐药细胞中表达。2.与鼻咽癌亲本细胞CNE-1比较,CK17在鼻咽癌紫杉醇耐药细胞CNE-1/Taxol中的表达显著降低,可能与鼻咽癌紫杉醇耐药相关。3. FOLR1在鼻咽癌细胞中与CK17存在蛋白质相互作用,可能通过影响其下游的信号通路,促进肿瘤细胞生长,抑制细胞凋亡,在鼻咽癌紫杉醇耐药中发挥重要作用。更多还原

【Abstract】 The folate receptor1(FOLR1) is a member of surface glycoprotein with a very high affinity for folates. In the previous study, we reported that FOLR1expression level was closely correlated to both proliferation of nasopharyngeal carcinoma (NPC) cells and taxol-resistant in NPC cells. In this study, both parental NPC cell lines and taxol-resistant NPC cell lines will be used as subjects, and we aim to disclose the mechanism of taxol resistance in NPC cells through investigating the protein interaction of FOLR1. Firstly, screening the interaction proteins of FOLRl using co-immunoprecipitation technique in which FOLR1will be used as a bait protein, and primarily identifying the common interacting proteins and differential interacting proteins of FOLR1between both parental and taxol resistant NPC cells,which is cytokeratin17(CK17). Secondly, the interacting proteins of FOLR1will be verified by co-immunoprecipitation assay and fluorescent subcellular co-localization techniques. Thirdly, we will depict the possible signal pathway of FOLR1, and build protein interaction network through the analysis of interacting proteins with known mechanism of action, and indepth investigation of interacting proteins with unknown function. Then, the FOLR1signal pathway will be confirmed through the detection on activation of FOLRl signal molecules caused by silencing of FOLRl or CK17. Based on the above results, the correlation will be further detected between taxol resistance and FOLR1signal pathway by inhibition of FOLR1or CK17signal molecules using RNA interference. The role that FOLR1playing in the mechanism of taxol resistance will be in detailed elucidated in nasopharyngeal carcinoma. 1Screening of the interaction proteins of FOLR1Objectives:To screen the interacting protein with FOLR1in human Taxol-resistant nasopharyngeal carcinoma cells(NPC).Methods:The interacting proteins of FOLR1were screened by co-immunoprecipitation and SDS-PAGE gel staining techiqiue in parental NPC and taxol-resistant NPC cells (CNE-1/Taxol, CNE-2/Taxol, HNE-2/Taxol), and identified by mass spectrometry analysis. Results:Compared with the control, there were5different bands on SDS-PAGE gel after electrophoresis of complexes which FOLR1antibody was co-immunoprecipitated with total CNE-2/taxol cell protein, and the molecular weight was about120kD,58kD,50kD,44kD,30kD,respectively;3differences bands were found on gel which FOLR1antibody co-immunoprecipitated with total CNE-1/taxo110cell protein, and the molecular weight was about60kD,55kD,50kD, respectively; Also,2different bands were detected on gel which anti-FOLR1antibody co-immunoprecipitated with CNE-1/taxol13cell protein, the molecular weight was about50kD,36kD, repectively. Combined with the3experimental results of different precipitation, a constand differential band showed on gel and its molecular weight was about50kD. It indicated that α～50kD protein on SDS-PAGE gel of FOLR1co-immunoprecipitation may be the proteins interacting with FOLR1. Through the flight mass spectrometry analysis of differential bands, it showed that the protein of differential bands was cytokeratin17.Conclusion:The FOLR1protein may be interacting with CK17protein in taxol-resistant NPC cells. 2Verification of Protein Interaction between FOLR1and CK17proteinsObjective:To verify the FOLR1protein interacting with CK17protein, which was screened by Co-immunoprecipitation and MACS analysis, and identified if both of them existing interactions under physiological conditions.Methods:Both parental NPC cells (CNE-1) and Taxol-resistant NPC cells (CNE-1/Taxol) were analyzed for protein interaction between FOLR1and CK17proteins by co-immunoprecipitation and Western blot using anti-FOLR1and Anti-CK17antibody; Co-localization of FOLR1and CK17proteins was determined by immunofluorescence assay; The known possible interacting proteins of FOLR1and CK17were retrieved and analyzed by using Visant biological information database network and its software.Results:1The protein expression of CK17can be detected in the protein complexes of co-immunoprecipitation of FOLR1antibodies with total protein of Taxol-resistant NPC cells (CNE-1/taxol).It was not detectable in negattive controls.2The protein expression of FOLR1can be detected in the protein complexes of co-immunoprecipitation of CK17antibodies with total protein of Taxol-resistant NPC cells (CNE-1/taxol).It was not detectable in NP69cells.3The FOLR1expression was observed in the cytoplasm and cell membrane, and a strong expression of CK17protein was observed in the cytoplasm by using immunofluorescence laser confocal microscopy. A fusion of both proteins in the cytoplasm was detected.4Analysis of Visant biological information network demonstrated that five proteins might be interacted with FOLR1protein (LYN, UBC, NCAPH2, IRAK3, CUL3), and sixteen proteins might be interacted with CK17proteins (UBC, YWHAQ, KRT8, KRT72, SNAPAP, KRT7, EGFR, CCDC85B, KRT6A, APC, GABARAPL1, GRB2, USP1, UCHL1, COPS5, SUM02). There is a common interacting protein for both FOLR1and CK17proteins, and it is Ubiquitin C.Conclusion:1There is a physical interaction between FOLR1and CK17 proteins in both parental NPC cells and Taxol-resistant NPC cells.2Ubiquitin C is a common interacting protein between FOLR1and CK17proteins. 3The functional interaction of FOLRl and CK17proteinsObjectives:To observe the expressional changes of FOLR1, CK17and related signal transduction genes after inhibition of FOLR1or CK17.Methods:The knock-down of genes was carried out by siRNA technique. mRNA and protein expression level of CK17and FOLR1genes were detected by real time PCR and Western blot in NPC cells (CNE-1) and taxol-resistant cells (CNE-1/Taxol). The expressional changes of signal transduction genes after FOLRl inhibition was conducted by cDNA microarray.Results:Expression of CK17mRNA and protein were decreased in Taxol-resistant NPC cells (CNE-1/Taxol) compared with parental cells. No significant change was observed in the expression of the FOLR1gene after knocked-down of CK17gene; However, mRNA and protein expression level of CK17gene was significantly down-regulated after the knocked-down of the FOLR1gene in taxol-resistant NPC cells (CNE-1/Taxol). cDNA Microarray analysis showed that, after FOLR1gene silencing, expression of5genes which known possible interacting with FOLR1, were down-regulated1.0-1.7times, and CK17expression was decreased2.5times.Conclusion:1.Cytokeratin17expression was detected in nasopharyngeal epithelial cells of human, parental NPC cells, and taxol-resistant NPC cells.2.Comparison with parental CNE-1cells, the expression of CK17were significantly decreased in taxol-resistant NPC cells, and it may be associated with Taxol-resistant nasopharyngeal carcinoma.3.The FOLR1protein can be interacting with CK17protein in NPC cells, and promote the growth of tumor cells, inhibit cell apoptosis, and play an important role in taxol resistance in NPC. The role may be playing through signaling pathway and its downstream effects.更多还原