鼻咽癌紫杉醇耐药中FOLR1相互作用蛋白的筛选及机制的初步探索

发布时间：2018-05-20 04:48

本文选题：鼻咽癌 + 紫杉醇耐药　；参考：《中南大学》2013年博士论文

【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。
[Abstract]:Folic acid receptor A (FOLR1) is a high affinity receptor for folic acid uptake by cells. Previous studies have found that the expression of FOLR1 is closely related to the occurrence of nasopharyngeal carcinoma and the taxol resistance phenotype. The taxol resistance phenotype of nasopharyngeal carcinoma caused by FOLR1 seems to be irrelevant to the function of folic acid transport. The taxol resistant cell line is the research object. From the interaction between proteins as the breakthrough point, the correlation between the FOLR1 pathway and the drug resistance of taxol in nasopharyngeal carcinoma is explored. First, the interaction proteins are screened by FOLR1 polyclonal antibody as bait by immunoprecipitation technology, and it is preliminarily identified in the taxol resistant cells of nasopharyngeal carcinoma. FOLR1's interacting protein, keratin 17 (Cytokeratin17, CK17), then identifies interaction proteins by immunofluorescence, Western Blot, immunofluorescence co localization, and determines the interaction between FOLR1 and CK17 in the parent and taxol resistant cells of nasopharyngeal carcinoma and taxol resistant cells. Through bioinformatics network The interaction mechanism of interaction protein is analyzed, and the possible pathway of FOLR1 is analyzed. It is found that there is a common interacting protein between the two - ubiquitin C (ubiquitin C, UBC). The interaction proteins of the two are involved in cell differentiation, cell cycle, DNA modification, signal transduction and so on. Then siRNA interference technique is used to silence FOLR1 or CK17. To detect the activation of the molecules of the pathway, we found that the FOLR1 gene was silenced and the expression of CK17 decreased in both the transcription and protein levels, while the expression of the CK17 gene was silent, and the expression of FOLR1 was not changed. It was analyzed that the taxol resistance phenotype of nasopharyngeal carcinoma caused by FOLR1 may be used in this pathway through the interaction between FOLR1 and CK17, and the interaction between the two might be possible. We infer that the increased expression of FOLR1 in taxol resistant cells in nasopharyngeal carcinoma may play a role in the induction and regulation of signal molecules associated with tumor cell growth or apoptosis by protein interaction. Figure 45, table 21, 87 references
Screening of 1FOLR1 interacting proteins
Objective: to screen the interaction protein of FOLR1 in paclitaxel resistant human nasopharyngeal carcinoma cells.
Methods: the interaction proteins of FOLR1 in normal nasopharyngeal epithelial cells (NP69) and three strains of nasopharyngeal carcinoma (CNE-1/Taxol, CNE-2/Taxol, HNE-2/Taxol) were screened by immunoprecipitation, SDS-PAGE electrophoresis and gel staining, and the interaction proteins were identified by mass spectrometry.
Results: after coprecipitation of FOLR1 antigen antibody complex and Protein A agarose beads, the protein of CNE-2/taxol cell was compared with the control, and there were 5 different bands. The molecular weight was about 120kD, 58kD, 50kD, 44kD, 30kD; CNE-1/taxol10 cell protein was electrophoretic after immunization. Compared with the control, there were 3 difference bands and molecular weight respectively. About 60kD, 55kD, 50kD; CNE-1/taxol13 cell protein electrophoresis after immunoprecipitation, compared with self control, there are 2 difference bands, the molecular weight is about 50kD, 36kD. combined with 3 immunoprecipitation results, the difference bands of -50kD are shown on different gels, and the protein of -50kD on SDS-PAGE gel may be interacting with FOLR1. The proteins were analyzed by flight mass spectrometry, and the results showed that the protein was cytokeratin17.
Conclusion: the FOLR1 protein in paclitaxel resistant human nasopharyngeal carcinoma may interact with CK17 protein.
Validation of the interaction between 2FOLR1 and CK17 protein
Objective: to verify the interaction protein CK17 of FOLR1 and identify the interaction between the two species under physiological conditions.
Methods: the interaction between FOLR1 and CK17 in nasopharyngeal carcinoma parent cells (CNE-1) and taxol resistant cells (CNE-1/Taxol) of nasopharyngeal carcinoma (CNE-1/Taxol) was examined by immunoprecipitation and Western blot method. The co localization relationship between FOLR1 and CK17 was analyzed by double immunofluorescence method, and the bioinformation network database was retrieved and analyzed by using Visant soft parts. Identify the known interacting proteins of FOLR1 and CK17.
Results: 1. FOLR1 antibody was used to immunoprecipitation of the total protein of taxol resistant cell CNE-1/taxol in nasopharyngeal carcinoma. The expression of CK17 could be detected in the complex, and the expression of CK17 was not detected in the negative control.
2. after the immunoprecipitation of the total protein of the taxol resistant cell CNE-1/taxol of nasopharyngeal carcinoma with CK17 antibody, the expression of FOLR1 was detected in the complex, and the expression of FOLR1 was not detected in the NP69 of the control cells.
The expression of FOLR1 protein in both cytoplasm and cell membrane was observed by 3. immunofluorescent laser confocal microscopy. The expression of CK17 protein in the cytoplasm and the fusion of the two proteins in the cytoplasm.
4. Visant software search of biological information network found that there are 5 interacting proteins of FOLR1, namely, LYN, UBC, NCAPH2, IRAK3, CUL3, CK17, respectively, 16 proteins have common interacting proteins. Mass, ubiquitin C (Ubiquitin C)
Conclusion: 1. there is a physical interaction between FOLR1 and CK17 in nasopharyngeal carcinoma cells and paclitaxel resistant cells.
2.FOLR1 and CK17 have a common interacting protein, ubiquitin C..
Functional interaction between 3FOLR1 and CK17
Objective: To observe the gene expression changes of signal transduction pathway after inhibiting FOLR1 or CK17 gene expression by RNA interference.
Methods: real time PCR and Western blot were used to detect the expression of CK17 in the parent cell (CNE-1) of nasopharyngeal carcinoma (CNE-1) and its taxol resistant cells (CNE-1/Taxol), and the expression of CK17 in CNE-1 cells was silenced by RNA interference technique, and the expression of the FOLR1 cells was observed and the changes of the genes were observed. Microarray technology was used to analyze the changes of mRNA expression in drug-resistant cells after FOLR1 gene silencing.
Results: the expression of CK17mRNA and protein in the taxol resistant cell CNE-1/Taxol of nasopharyngeal carcinoma was markedly weakened, and the expression of FOLR1 gene was not significantly changed after the CK17 gene expression of CNE-1 in nasopharyngeal carcinoma cells was reduced. However, the mRNA and protein expression level of the CK17 gene was reduced after the FOLR1 gene of the taxol resistant cell CNE-1/Taxol of nasopharyngeal carcinoma was reduced. A significant downregulation of.CDNA Microarray gene chip showed that after the FOLR1 gene was silenced, the expression of 5 genes interacting with FOLR1 were all down regulated by 1.0-1.7 times, and the expression of CK17 was down to 2.5 times.
Conclusion: 1. Cytokeratin17 is expressed in human immortalized nasopharyngeal epithelial cells, NPC cells and nasopharyngeal carcinoma paclitaxel resistant cells.
2. compared with the parent cell CNE-1 of nasopharyngeal carcinoma, the expression of CK17 in the taxol resistant cell CNE-1/Taxol of nasopharyngeal carcinoma was significantly decreased, which may be related to the taxol resistance of nasopharyngeal carcinoma.
3. FOLR1 has protein interaction with CK17 in nasopharyngeal carcinoma cells, which may play an important role in the taxol resistance of nasopharyngeal carcinoma by affecting the downstream signal pathway, promoting the growth of tumor cells and inhibiting apoptosis.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.63

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