Preliminary Study On The Changes Of Lymph Node Metastatic Protein Profile In PTMC Based On ITRAP/TMT-Labeling Proteomics

Objective: To analyze the differentially expressed proteins(DEPs)between papillary thyroid microcarcinoma(PTMC)with lymph node metastasis and PTMC without lymph node metastasis using iTRAQ/TMT-based quantitative proteomics technology.Bioinformatics techniques were used to analyze the biological processes and signal pathways involved in these proteins and to screen potential protein biomarkers.It provides a theoretical basis for the preliminary elaboration of the pathogenesis of lymph node metastasis of PTMC and the search for clinical diagnostic indicators and therapeutic targets.Methods: Six patients with PTMC who underwent initial surgical treatment at the Thyroid Surgery Division in China-Japan Union Hospital of Jilin University were selected as research objects.Intraoperative specimens of tumor tissue(T)and paracancerous tissue(N)were collected.According to whether lymph node metastasis occurred in the results of postoperative paraffin pathology,the patients were divided into two groups: without lymph node metastasis group(N0)and lymph node metastasis group(N1).iTRAQ/TMT technique combined with LC-MS/MS technique was used for qualitative and quantitative analysis of proteins in tissues.The identified differentially expressed proteins were analyzed by GO,KEGG pathway,PPI and other bioinformatics.The bioinformatics analysis results and literature reports were integrated to screen meaningful proteins for further validation.Results:(1)Overview of protein identification: Based on iTRAQ/TMT proteomic analysis,a total of 487 up-regulated DEPs and 486 down-regulated DEPs were identified in T/N group.A total of 432 DEPs were identified in the N0-T/N0-N group,of which 205 were up-regulated and 227 were down-regulated.In the N1-T/N1-N group,502 DEPs were identified,242 of which were up-regulated and 260 of which were down-regulated.(2)GO annotation analysis: all three groups of DEPs were mainly involved in metabolic process,biological process regulation,cellular process,response to stimulation and other biological processes;Mainly play binding,enzyme regulation activity,catalytic activity and other molecular functions;It is mainly located in extracellular regions,organelles,etc.(3)KEGG pathway enrichment and cluster analysis: In T/N group,differentially expressed proteins were mainly enriched in Thyroid hormone synthesis,Cholesterol metabolism,Nitrogen metabolism and other signaling pathways.In N0-T/N0-N group,DEPs were mainly enriched in the Thyroid hormone synthesis,Tyrosine metabolism and Thyroid cancer pathways.In N1-T/N1-N group,the more significant enrichment pathways were Nitrogen metabolism,Cholesterol metabolism,Lysosome,Ribosome,etc.(4)According to the results of bioinformatic analysis,Cholesterol metabolism pathway may play an important role both in the pathogenesis and LNM of PTMC.DEPs located in this pathway include NPC2,VDAC1,Apo E,Apo B,Apo C3,LAL and other proteins.Among them,NPC2,VDAC1,Apo E,Apo B and Apo C3 proteins may be involved in the occurrence and development of tumors.The significant down-regulation of NPC2,VDAC1 and LAL between N1 group and N0 group suggesting that they may be closely related to lymph node metastasis of PTMC.(5)Screenings of differentially expressed proteins: Besides proteins enriched in the cholesterol metabolism pathway,we found that ANXA1,PDLIM4,FN1 proteins were closely related with LNM of PTMC,through literature research,bioinformatics analysis,and combining with Venn diagram analysis.And these proteins provide a certain reference for the study of physiological and pathological mechanisms of LNM and screening potential biomarkers of PTMC.Conclusion:(1)The iTRAQ/TMT labeling quantitative proteomic analysis demonstrates that the differentially expressed proteins are mainly involved in cellular process,single-organism process,biological process,metabolic process and response to stimulus,which are potentially associated with the development and LNM of PTMC.(2)Cholesterol metabolism pathway may have an important correlation with the pathogenesis and LNM of PTMC.NPC2,VDAC1,Apo E,Apo B,Apo C3,and LAL are DEPs located in this pathway,and the specific mechanism of this pathway in the lymph node metastasis of PTMC is of high research value.(3)NPC2,VDAC1,LAL,ANXA1,PDLIM4,FN1 and other proteins are closely related to lymph node metastasis in PTMC,and these proteins provide a certain reference for the study of pathological mechanism of lymph node metastasis and screening potential biomarkers.