The Biological Functions And Clinical Applications Of NPC2 In Thyroid Cancer Based On Proteomic Sequencing

BACKGROUND:Papillary thyroid carcinoma(PTC)is the most common pathological type of thyroid cancer.In recent years,the incidence rate has gradually increased,and the precise diagnosis of PTC and the selection of appropriate individualized treatment modalities are currently a greater challenge for clinicians.Protein is an important material basis for life activities and is the main performer of cell proliferation,differentiation,aging and death.Unlike genomics,transcriptomics and metabolomics,proteomics takes proteins as the object of study and elucidates the underlying molecular mechanisms by analyzing the changes in protein levels.Studies have shown that,in addition to neurological diseases such as Alzheimer’s disease,it may play a regulatory role in the development of gastric cancer and glioma,but its role and molecular mechanism in thyroid cancer are unclear.METHODS:First,tissue specimens from PTC patients who underwent initial surgery were collected from the Department of Thyroid Surgery,and proteomic sequencing was performed by i TRAQ-TMT-based mass spectrometry.A series of bioinformatics methods such as KEGG,Mfuzz and PPI were used to screen for potentially clinically significant DEPs.biomarkers with certain diagnostic efficacy were identified by machine learning and Nomogram methods after PRM validation.based on TIMER,EPIC,QUENTISEQ,STRING,Gene MANIA,WGCNA and other methods for exhaustive bioinformatics analysis of the pan-cancer analysis.In addition,q PCR,Western blot,CCK-8,flow cytometry,Transwell assay,and immunofluorescence were used to validate the biological function of NPC2 in TPC-1 cell model.Finally,the sample size was further expanded to collect clinical specimens and correlation between NPC2 expression and clinicopathological features of PTC patients was assessed by q PCR and IHC methods.RESULTS:1.Proteomics sequencing and machine learning-based probing of thyroid cancer biomarkersFirst,based on protein sequencing results,a total of 5203 DEPs were identified from the tumor-normal comparison group or the N1-N0 comparison group,respectively.mfuzz analysis showed that the signaling pathways enriched by DEPs in each comparison group,from paraneoplastic,lymphatic metastasis-free thyroid cancer to thyroid cancer with lymphatic metastasis,were closely associated with thyroid cancer disease progression.PPI analysis showed that these The results of PPI analysis showed that these DEPs were mainly enriched in the following metabolism-related signaling pathways: tricarboxylic acid cycle,PI3K-Akt pathway,apoptosis,cholesterol metabolism,pyruvate metabolism,and thyroid hormone synthesis.Further,the sample size was expanded to collect tissue specimens from the validation cohort(20 pairs),and the PRM validation results showed that 18/20 target proteins were consistent with the sequencing results,and the expression levels of some of the target proteins correlated with PTC lymph node metastasis.Machine learning of the validated DEPs showed that PDLIM4,ANXA1,PKM,NPC2 and LMNA were able to distinguish benign and malignant thyroid nodules well,while FN1 was moderately effective in identifying the presence of lymph node metastasis with an AUC of 0.690.Finally,based on the TCGA database,the following five PRM-validated proteins were associated with PTC prognosis FN1,IDH2,VDAC1,FABP4,and TG,and the Nomogram model built from them was able to predict prognosis well with a consistency index of 0.685(confidence interval: 0.645-0.726).2.Exploring the role of NPC2 in thyroid cancer and pan-cancer based on bioinformatics analysis methodTo further explore the molecular mechanism of lymph node metastasis in thyroid cancer,we first divided the patients in TCGA-THCA database into N0 and N1 groups,and screened DEGs with Fold-change>1.3 or <0.7 as cut-off values.the results of WGCNA analysis showed that black module was associated with the development of PTC,and this gene set of DEGs were in good agreement with PTC progression,mainly including: NPC2,KLHDC8 A,METTL7B,ETV4,LRP4,LIPH,etc.Based on the above series of studies,we took NPC2 as the main object of the follow-up study.Systematic bioinformatics analysis of thyroid cancer was performed against NPC2 in TCGA and GTEx databases.The results showed that NPC2 expression was higher in PTC compared with paraneoplastic tissues,and higher in Stage III and Stage IV stages than Stage II,and low expression in PTC was associated with high OS.Further,we performed a multi-tumor pan-cancer bioinformatics analysis of NPC2 in TCGA,GTEs,CPTAC and c Bio Portal databases.The results showed that NPC2 was differentially expressed in multiple tumors and correlated with tumor survival(OS).Importantly,analysis in immune-related databases such as TIMER,EPIC,and QUENTISEQ showed that NPC2 was mainly positively correlated with MHC and immunostimulatory factors.Genes interacting with NPC2 were mainly enriched in lysosomal,cholesterol metabolism,autophagy and other pathways.It is suggested that NPC2 may regulate the development and prognosis of tumor through the immune microenvironment.3.Biological functions of NPC2 in thyroid cancer developmentTo further verify the biological function of NPC2 in thyroid carcinogenesis,we selected TPC-1,a papillary thyroid cancer cell line with high NPC2 expression,as the main study target.First,sh RNA technology was applied to construct a cell model in TPC-1.The results showed that all three plasmids significantly reduced NPC2 expression at the m RNA level;while at the protein level,the knockdown rate of sh NPC2-1 was up to more than 70%,so this cell model was selected for subsequent functional experiments.the results of CCK-8 showed that NPC2 knockdown significantly reduced the cell proliferation ability of TPC-1 compared with sh NC.This inhibitory effect persisted when cisplatin and rapamycin pretreatment was administered.Apoptosis results based on flow cytometry assays showed that NPC2 knockdown increased the level of cisplatin-induced apoptosis but inhibited the level of Rapainduced apoptosis compared to the sh NC group.Cell cycle results showed that cisplatin induced a significant decrease in G2/M phase block compared to the sh NC group,whereas NPC2 knockdown significantly increased cisplatin-induced G2/M phase block.Transwell results showed that NPC2 knockdown significantly inhibited the cell migration and cell invasion ability of TPC-1 compared with the sh NC group.These results suggest that NPC2 may be involved in the regulation of cell proliferation,apoptosis,cell cycle,cell migration and invasive ability in thyroid cancer.4.Correlation between NPC2 and clinicopathological characteristics of thyroid cancer patientsTo further verify the correlation between NPC2 expression and clinicopathological characteristics,we recollected thyroid cancer tissue specimens and basic clinical data from PTC patients(N=47 cases).The NPC2 protein expression level was observed by IHC and its m RNA expression level was detected by applying q PCR,respectively.q PCR results showed that NPC2 was highly expressed in cancer tissues compared with para-cancer tissues.The ROC curve was plotted to assess the diagnostic efficacy of NPC2 for benign and malignant thyroid nodules,and the results showed that the AUC=0.875,95% CI: 0.754-0.996.IHC results showed that NPC2 was highly expressed in cancerous tissues compared with paraneoplastic tissues.The IHC scores of cancer and paraneoplastic tissues from the same patient were compared,and Ratio≥ 2 was defined as a relatively high expression group,and vice versa,as a relatively low expression group.The results showed that the tumor diameter of PTC was longer and calcification was more likely to appear within the tumor in the NPC2 relative high expression group compared with the NPC2 relative low expression group.It is suggested that the expression of NPC2 may be related to the clinicopathological characteristics of thyroid cancer patients.CONCLUSION1.Biomarkers based on proteomics and machine learning to explore thyroid cancer(1)At the protein level,the development of thyroid cancer may be associated with the following metabolic signaling pathways: tricarboxylic acid cycle,PI3K-Akt pathway,cholesterol metabolism,pyruvate metabolism and thyroid hormone synthesis.(2)Based on machine learning screening,the following proteins can be used as potential markers for the diagnosis of benign and malignant thyroid: PDLIM4,ANXA1,PKM,NPC2,LMNA and FN1.(3)Nomogram model based on five proteins(FN1,IDH2,VDAC1,FABP4 and TG)can better predict the prognosis of thyroid cancer patients.2.Exploring the role of NPC2 in thyroid cancer based on bioinformatics analysis methods(1)Based on WGCNA analysis,black module is associated with the development of thyroid cancer,and the main genes include: NPC2,KLHDC8 A,METTL7B,ETV4,LRP4,LIPH,etc.(2)Based on bioinformatics analysis method,we confirmed that NPC2 was highly expressed in thyroid cancer tissues and correlated with OS.The genes interacting with NPC2 were mainly enriched in lysosomal,cholesterol metabolism,autophagy and other pathways.(3)NPC2 was differentially expressed in pan-cancerous tissues of different tumors and correlated with survival(OS)in a variety of tumors.It may regulate the development and prognosis of thyroid cancer through the immune microenvironment.3.Biological functions of NPC2 in thyroid carcinogenesis and development(1)NPC2 is highly expressed in most thyroid cancer cell lines.TPC-1 was selected as the main target and a cell model with NPC2 knockdown was successfully established.(2)NPC2 knockdown significantly inhibited the cell proliferation ability of TPC-1,inhibited the increase of apoptosis level induced by Cis and Rapa,inhibited the G2/M phase block,and also significantly inhibited the cell migration and cell invasion ability of TPC-1.4.Correlation analysis of NPC2 and clinicopathological features of thyroid cancer(1)In the tissue specimens of the validation cohort,the high expression of NPC2 in the tumor tissues was confirmed at the m RNA level and protein level,respectively.(2)The relatively high expression of NPC2 may be correlated with clinicopathological features such as tumor diameter and tumor calcification.