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Objective:Using Isobaric tags for relative and absolute quantification (iTRAQ) quantitative proteomic technique combined with two-dimensional liquid chromatography tandem mass spectrometry (2DLC-MS/MS) method we screen nasopharyngeal carcinoma metastasis related proteins, respectively validate these proteins at the level of gene and protein, so as to screen out predicting metastasis of nasopharyngeal carcinoma (NPC) molecular markers, for researching nasopharyngeal carcinoma invasion metastasis mechanism to provide scientific theoretical basis, these molecular markers in clinical can predict the prognosis and survival rate of nasopharyngeal carcinoma, finally guide the rational treatment of nasopharyngeal carcinoma.Methods:cultivate non-metastatic nasopharyngeal carcinoma cell line (6-10B) and high metastatic nasopharyngeal carcinoma cell line (5-8F), extract proteins respectively, measure of protein concentration. iTRAQ combined with2DLC-MS/MS was used to separate and identify different expressing proteins of two cell lines, two technology repeated. Using ProteinPilotTM4.2search and identification transfer relevant proteins in Swissprot protein database, selected transfer relevant proteins to meet the requirements for Biological information analysis. Bioinformatics analysis results for reference,Through the analysis of the uniprot software and related literature retrieval,Picked out23target proteins for qRT-PCR (Quantitative Reverse transcriptase Polymerase chain reaction), detect the mRNA level of target proteins. Western Blot was used to verify the protein expression level in the two kinds of cells.Immunohistochemistry was used to verify these proteins’expression differences between the10cases without cervical lymph node metastasis of nasopharyngeal carcinoma tissues and20cases of neck lymph node metastasis of nasopharyngeal carcinoma tissues.Using SPSS17.0statistical software for data analysis.Results:According to the selection criteria,We have screened190nasopharyngeal carcinoma metastasis related proteins. Using GO analysis found that these different proteins was mainly located in cytoplasm (26%) and the nucleus (22%), the biological processes involved in contained signal transduction, metabolism, oxidation reduction, transcription, translation, transport, protein folding, cell movement, etc. molecular function involved in had protein binding, nucleotide binding, calcium ion binding, ribosome structures, oxidation reduction activity, etc. Using KEGG pathway analysis,we found that these proteins involved in many cancer related signal pathways, such as:cell cycle, apoptosis, MAPK signal pathway, p53signaling pathway and so on. PPI protein interactions network showed the nodes proteins were mainly involved in cell cycle regulation, cell growth, proliferation, apoptosis and aging, signal transduction,protein degradation, metabolism, transport and glycosylation modification, redox balance and so on. qPCR results show eleven genes (ADAMTSL4、DIABLO、PDIA4、CALR、SPTBN1、PPIA、GSN、 SQSTM1、TXN、RAN、TRIM29) the condition of mRNA expression were consistent with iTRAQ results, the expression level of ADAMTSL4、DIABLO、PDIA4、CALR and SPTBN1were higher in5-8F than in6-10B,On the contrary,the expression lever of PPIA、GSN、 SQSTM1、TXN、RAN and TRIM29were lower in5-8F than in6-1OB The expression level of mRNA of three gene (ANXA5, PTRF, NDRG1) had no difference between the5-8F and6-1OB, mass spectrometry results show that the protein expression level is lower in the5-8F, in addition, there are nine genes (NPM1, VCP, RDX, PTGES3, MT2A, EIF4EBP1, LGALS3BP, GPX1and AIMP1),their mRNA expression level were contrary to the mass spectrometry results.Western blotting test found that the expression levels of RAN and TRIM29were significantly higher in the5-8F than6-10B, the expression levels of ANXA5and SQSTM1were significantly lower in the5-8F than6-10B.us ing Immunohistochemistry verified that the expression level of RAN and TRIM29in metastatic nasopharyngeal carcinoma tissues were significantly higher than in tissues without metastasis, on the other hand, the expression trend of ANXA5and SQSTM1was opposite.Conclusions:1、use the high transfered nasopharyngeal carcinoma (NPC) cell line5-8F and nontransfered nasopharyngeal carcinoma cell line6-10B as the research model, for the first time by iTARQ combining2DLC-MS/MS method selected for metastasis related differentially expressed proteins altogether190.GO function analysis and KEGG pathway analysis shows the biological functions and the signaling pathways these proteins involved in, offers clues for nasopharyngeal carcinoma invasion metastasis research. PPI network diagram construction for nasopharyngeal carcinoma metastasis research provides large amount of information and new ideas.2、ADAMTSL4, DIABLO/smac, PDIA4, CALR, SPTBN1expressed lower in5-8F than in6-10B, PPIA, GSN, SQSTM1, TXN, RAN, TRIM29expressed higher in5-8F than in6-10B, instructed that these genes might be closely related to invasion and metastasis of nasopharyngeal carcinoma cells.3、we verify respectively in cell and tissue level the four genes RAN, TRIM29, ANXA5, SQSTM1is closely related to the invasion and metastasis of nasopharyngeal carcinoma (NPC), may be the important molecular markers predicting metastasis and prognosis of nasopharyngeal carcinoma (NPC).For the treatment of nasopharyngeal carcinoma (NPC) provides a broad prospect, its specific mechanism and clinical application value remains to be further research.14Figures,6tables,81references
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