| Part One SCREENING THE BIOMARKER OF RECURRENT NASOPHARYNGEAL CARCINOMABackgroud and ObjectiveNasopharyngeal carcinoma(NPC)is one of the most common cancer in southern China,such as Guangxi,Guangdong,Fujian provinces.As far as we know,radiotherapy(RT)is the main treatment for NPC.Due to its special location and normally clinical manifestations,NPC is rare for early diagnosis.Unfortunately,approximately 10-15% of NPC patients still have local recurrence after the effective therapies.After a re-radiotherapy,both the effect and prognosis of the treatment were poor.The pathogenesis of recurrent nasopharyngeal carcinoma(r NPC)is unknown now,most researches suggested that the radiation resistance is one of the main reason of r NPC.Further more,there is no useful biomarkers which can be used for monitoring,diagnosis,treatment of r NPC in clinic.Therefore,it is an urgent need for identifying new putative biomarkers of r NPC.In this study,we used tandem mass tags(TMT)labeling and HPLC fractionation followed by liquid chromatograph-tandem mass spectrometry(LC-MS/MS)analysis to identify differentially expressed proteins,try to picture a tree of proteins related to r NPC.MethodWe collected all samples following the standard of inclusion criteria strictly.Without any treatment,we collected the blood.r NPC patients and non-r NPC patients were randomly assigned to two teams(A and B),each team included 10 recurrent serums and 10 no-recurrent serums.We used tandem mass tags(TMT)labeling and high performance liquid chromatography(HPLC)fractionation followed by liquid chromatograph-tandem mass spectrometry(LC-MS/MS)and Mascot search engine(v.2.3.0)software analysis to identify differentially expressed proteins,quality control of mass spectrometry was carried out.Reproducibility analysis of two repeated trials by scatter diagram was performed.Intensive bioinformatics analysis was then carried out to annotate those quantifiable targets,including protein annotation,functional classification,functional enrichment,functional enrichment-based cluster analysis,etc.Results(1)In total,the serum identified 635 proteins,among which 413 proteins were quantified.When setting quantification ratio of >1.2 as up-regulated threshold and <0.83 as down-regulated threshold.Down-regulated and up-regulated proteins between test1(recurrence(n 1= 10)VS no-recurrence(n2= 10))and test2(recurrence(n 3= 10)VS no-recurrence(n 4= 10))were 41 and18 respectively.Quality control of mass spectrometry was qualified.According to the scatter diagram and Pearson Correlation Coefficient,the result of reproducibility analysis was qualified.(2)GO Classification of Terms Level 2The up-regulated proteins were main involved in biological process of single-organism process(26),cellular process(25),response to stimulus(20)etc,and the cellular component main included cell(23),organelle(21),macromolecular complex(13)etc,molecular function main included binding(15),catalytic activity(11)etc.The down-regulated proteins were main involved in single-organism process(40),biological regulation(39),response to stimulus(35)etc,the cellular component main included extracellular region(37),cell(34),membrane(20)etc,molecular function main included binding(15),catalytic activity(11)etc.(3)GO-based Enrichment AnalysisThe up-regulated proteins molecular function were main enriched in guanyl ribonucleotide binding,GTP binding function,etc,and the cellular component main included microtubule cytoskeleton etc.In addition,numerous proteins were involved in response to microtubule-based movement,cell division biological process etc.The down-regulated proteins the cellular component were main enriched in nucleosome,DNA bending complex,chromatin,etc,and molecular function main included antigen binding,ATPase activity,etc,In addition,numerous proteins were involved in nucleosome assembly,protein-DNA-complex assembly biological process etc.(4)KEGG Pathway EnrichmentKEGG Pathway Enrichment included hsa05034 Alcoholism-Homo sapiens(human),hsa04970 Salivary secretion-Homo sapiens(human),hsa05144 Malaria-Homo sapiens(human)pathways,According to KEGG pathway analysis,the enrichment ratio of hsa05034 Alcoholism-Homosapiens(human)pathway is highest,moreover,there are 4 dis-regulated proteins(CALM,H2 B,H3,H4)involved in this pathway.Further,we did a research about these dis-regulated proteins(CALM,H2 B,H3,H4),which shown that CALM was related to Cell proliferation,apoptosis,autophagy and cancer stem cells,DNA damage,cell movement etc.(5)Domain EnrichmentProtein domain enrichment analysis of dis-regulated proteins was found enriched in immunoglobulin-like domain,immunoglobulin-like fold etc.(6)Cluster AnalysisThe first cluster included 19 proteins(A2M,ACTN4,ALB,ALDOA,APP,CFD,F13A1,FN1,HRG,KNG1,PF4,PLG,PPBP,SERPINA1,SERPINE1,SERPING1,TIMP1,VWF),among which APP and SERPINE were up-regulated,the others were down-regulated;In the second cluster,there were APCS,APOA1,APOA4,CST3,F2,FGA,GSN,LTF,SERPINC1,TTR proteins,among which APCS,FGA,SERPINC1 were up-regulated,APCS,FGA,SERPINC1 proteins were down-regulated proteins.In the third cluster,there were APOA2,APOB,APOC3,APOE,LCAT,LPL proteins,among which only APOA2 was dis-regulated proteins(up-regulated).(7)Protein-protein Interaction(PPI)NetworkAll identified protein name identifiers were searched against the STRING database version 9.1 for PPI.According to the results by STRING,we found the high connetcive degree of disregulated proteins(ALB(degree=42);APP(degree=34),KNG1(degree=27);F13A1(degree=25);SERPING1(degree=25);A2M(degree=24);SERPINA1(degree=22);FGA(degree=19);SERPINC1(degree=16);APCS(degree=10);GSN(degree=10);APOA2(degree=9)).Conclusion1.We preliminary pictured a protein tree of r NPC,which included CALM,A2 M,APP,ALB,F13A1,KNG1,SERPINA1,SERPINE1 etc;2.CALM,A2 M,APP,ALB,F13A1,KNG1,SERPINA1,SERPINE1 could be the potential biomarkers for r NPC.Part Two USING ELISA TO CONFIRM THE MASS RESULTSObjectiveIn this part,we choosed CALM,APP,A2 M as the target proteins to confirm in using ELISA.We tried to elucidate the potential molecular mechanisms that are dysregulated and contributed to the pathogenesis of r NPC.Method Recolletced new serum samples,we used ELISA kits of CALM,APP,A2 M proteins to confirm the mass results.All statistical analyses were performed using the SPSS 21.0 software and Graphpad prism5.0,The differences between the two states of NPC were analyzed using a Mann-Whitney U test.In addition,the target protein were evaluated for its capacity to distinguish r NPC and nr NPC by makeing their ROC curves based on the ELISA scores.All results were considered to be significant when p-values were ?0.05.Results2.1 CALMFor the reason that the concentration of CALM were not normally distributed,we choose the medianand quartile method to describe the protein co ncentration.We found CALM significant difference between patients with r NPC compared to nr NPC in the levels of serum(p=0.0233),and the median concentration of CALM were 114.2ng/l and 45.10ng/l respetcively,the number of 25th% concentration were 13.27ng/l and40.03 ng/l respetcively,the number of 75th% concentration were 61.86ng/l and151.92 ng/l respetcively,which was in agreement with our MS results.Furthermore,we performed a ROC curve to evaluate the predictive value ofserum CALM,the area under the ROC curve is 0.6931(P=0.0233).The best cut-off point is 48.45ng/l,at which the sensitivity is 82.8%,the specificity is55%.2.2.APPFor the reason that the concentration of APP were not normally distributed,we choose the medianand quartile method to describe the protein co ncentration.We found APP significant difference between patients with r NPC compared to nr NPC in the levels of serum(p=0.044),and the median concentration of APP were 20.08ng/ml and 5.502ng/ml respetcively,the number of 25th% concentration were 4.561ng/ml and4.508ng/ml respetcively,the number of 75th% concentration were28.90ng/ml and 10.26ng/ml respetcively,which was in agreement with our MS results.Furthermore,we performed a ROC curve to evaluate the predictive value of serum APP,the area under the ROC curve is 0.666.The best cut-off point is 16.95ng/ml,at which the sensitivity is 55.2%,the specificity is 90.9%.2.3 A2MFor the reason that the concentration of A2 M were not normally distributed,we choose the medianand quartile method to describe the protein co ncentration.We found A2 M significant difference between patients with r NPC compared to nr NPC in the levels of serum(p=0.0015),and the median concentration of A2 M were 34.79 ng/ml and 75.67 ng/ml respetcively,the number of 25th% concentration were 13.27ng/ml and40.03ng/ml respetcively,the number of 75th% concentration were61.86ng/ml and 151.92ng/ml respetcively,which was in agreement with our MS results.Furthermore,we performed a ROC curve to evaluate the predictive value of serum A2 M,the area under the ROC curve is 0.757(P=0.0015).The bestcut-off point is 35.21ng/ml,at which the sensitivity is 56.5%,the specificity is86.7%.ConclusionCALM,A2 M,APP could be the potential biomarkers for r NPC. |
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