Screening Of Interaction Proteins Of FOLR1and Preliminary Study Of Its Mechanism In Taxol-resistance Nasopharyngeal Carcinoma Cells

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.