| Background:Thyroid carcinoma(TC)is the most common malignant tumor of the endocrine system,and the incidence rate is increasing year by year.TC is one of the fastest growing malignant tumors in the world and in China,including thyroid papillary.Papillary thyroid carcinoma(PTC)is the most common type of histology.Currently,PTC-related gene markers for detecting and evaluating prognosis include BRAF V600 E gene,RAS gene mutation and TERT promoter mutation.The protein markers include Galectin-3,Cytokeratin-19(CK19)and mesothelioma-associated antibody-1(HBME-1)and the like.In recent years,the development of proteomics has been providing more possibilities for an in-depth study of molecular markers of PTC,and for exploring the pathogenesis of PTC,defining diagnosis,and discovering new targets for treatment.Objectives:1.To screen differentially expressed proteins in thyroid papillary carcinoma(PTC)and benign thyroid nodules(BTN);2.To verify the effect of differential protein genes on PTC cell proliferation and find new targets related to thyroid cancer cell proliferation;3.To detect the expression of the target protein in PTC tissues and investigate its relationship with clinical pathological parameters.Methods:1.In total 36 fluid samples obtained through the fine needle puncture of percutaneous thyroid were selected in the study.Group A(1-3)includes 18 samples of BTN,and group B(1-3)include 18 PTC Samples.Label-free quantification(LFQ)proteomics method was used.The B/A differential protein group,functional annotation and pathway analysis of differentially expressed proteins by bioinformatics.2.The protein data with B/A up-regulation was analyzed.Twenty genes were selected for cell proliferation experiment screening.The small interfering RNA(siRNA)technology was used to knock down the expression of eachof the 10 target genesin K1 cell line.The effects of the loss-of-function of the target gene on the growth and proliferation of thyroid K1 cells were examined by Celigo cell counting and MTT assay.3.The expression of CAP1 and UGP2 in PTC,BTN,and normal thyroid tissue(NT)were detected by immunohistochemistry.The correlation between the expression of CAP1 and UGP2 and the clinicopathological parameters of PTC patients was analyzed.Results:1.In this study,Label-free method was used to separate and identify 10,619 peptides in BTN and PTC,and the total number of proteins was 1439.Annotated analysis of GO function of differentially expressed proteins revealed that the major cellular components of these proteins were mainly attributed to cells(13.3%),cell parts(13.3%),extracellular regions(12.3%),extracellular region pan(12.0%),macromolecular complex(6.7%),and membrane(6.8%).The molecular functions of the proteins mainly include binding(49.1%),and other catalytic activity(25.8%),molecular function regulation(8.0%),and structural molecular activity(5.5%).KEGG pathway annotation shows that PTC differential proteins affect many metabolic pathways in vivo,mainly include carbon metabolism and amino acid biosynthesis,glycolysis/gluconeogenesis,regulation of actin cytoskeleton,regulation of actin cytoskeleton,endoplasmic reticulum protein processing,protein processing,endoplasmic reticulum,connection of tight junction,carbon fixation in photosynthetic organisms,antigen processing and presentation,complement and coagulation cascades,and endocytosis.Protein differential analysis PTC/BTN(B/A)group identified 218 differentially expressed proteins,including 190 up-regulated proteins and 28 down-regulated proteins.2.The gene knockdown in human PTC K1 cell line was conducted by lentiviral vector infection.Twenty genes are screened for PTC proliferation by Celigo cell counting and MTT assay.The results show that the proliferation of cells after CAP1,UGP2,CAPG,CNDP2,ANP32 B gene knockdown was significantly less than that without gene knockdown.On the fifth day of culture,the shCtrl proliferation(5.94±0.27)times,the proto-oncogene shPC proliferation(2.46±0.05)times,the Fold change=2.41,and the shCAP1 proliferation only(2.28).±0.1)times,Fold change=2.6,hUGP2 only proliferated(2.55±0.09)times,Fold change=2.33,shCAPG proliferation(3.5±0.22)times,Fold change=1.7,shCNDP2 proliferation(3.73±0.13)times,Fold change =1.59,shANP32 B proliferation(3.86 ± 0.13)times,Fold change=1.54.3.Immunohistochemistry showed that UGP2 protein expressionwas positivein69.63%(94/135)of PTC tissues,in 37.5%(15/40)of BTN,in 35%(14/40)of NT,There was a significant statistical difference for the CAP1 expression between PTC and other tissues(P<0.05).CAP1 protein expressionwas positive in 77.04%(104/135)of PTC tissues,32.5%(13/40)of BTN,30%(12/30)of NT.There was a significant statistical difference for CAP1 expression between PTC and other tissues(P<0.05).The expression levels of UGP2 proteins were correlated with clinical stage and tumor size of patients with tumors(P<0.05),The expression levels of CAP1 proteins were correlated with clinical stage and lymphatic metastasis of patients with tumors(P<0.05),but not with age and gender.Conclusion:1.218 differentially expressed proteins were identified in thyroid nodule puncture fluid of PTC patients and BTN patients,including 190 up-regulated proteins and 28 down-regulated proteins.These proteins may have the potential to become PTC markers.2.Knockdown of CAP1,UGP2,CAPG,CNDP2,ANP32 B gene resulted in the inhibition of PTC cell proliferation,suggesting that these genes may be related to the proliferation of thyroid cancer cells,laying the experimental foundation for elucidating the pathogenesis and targeted therapy of PTC,offering a new perspective for PTC targeted gene therapy.3.CAP1 and UGP2 protein expression is significantly up-regulated in PTC,and associated with PTC lymph node metastasis,which may be used as a marker for diagnosis and prognosis in PTC and applied to the clinic. |
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