Screening And Identification Of Key Biomarkers Based On Transcriptome Sequencing And Expression Profile Analysis Of Papillary Thyroid Carcinoma

Objective:To explore the potential molecular pathogenesis of PTC and biotherapeutic targets preliminarily,the RNA sequencing(RNA-seq)technology was used to obtain the differential expression levels of papillary thyroid cancer(PTC)transcriptomes in northeastern Sichuan.Methods:(1)Collecting surgical tissue samples(including tumor tissues and normal thyroid tissues adjacent to the tumor)and related clinicopathological data of patients confirmed by pathology at the thyroid breast surgery in Affiliated Hospital of North Sichuan Medical College.(2)RNA-seq technique was used to analyze the differences in transcriptome expression between 20 PTC tumor tissue samples and 5 normal tumor samples adjacent to tumor.The differentially expressed genes(DEGs)were identified,and Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed to analyze and explore the function of differential genes.(3)Separately screening out the five genes with the most significant difference in up-and down-regulation,and using Real-time quantitative polymerase chain reaction(RT-RTqPCR)method to verify the mRNA expression level of 32 groups of PTC samples;meanwhile,the GEPIA public database was used to verify the mRNA expression level of the 10 genes.(4)Using STRING(Search Tool for the Retrieval of Interacting Genes)and Cytoscape software for module analysis to construct protein-protein interaction network(PPI)for genes with significant expression differences,and screen out the most significant modulesKey genes.(5)The RT-qPCR method was used to verify the expression of key genes in papillary thyroid cancer cell line BCPAP and normal thyroid cell line Nthy-ori-3-1;at the same time,analyzing the statistical correlation between it and the clinical pathological parameters of the previous tissue samples,drawing the ROC curve;combined with Cancer Genome Atlas(TCGA),Encyclopedia of Cancer Cell Lines(CCLE)and Oncomine database for biological information analysis to predict its potential prognostic value in PTC.Result:(1)In this study,3202 differentially expressed genes were identified,including 1772 up-regulated genes and 1430 down-regulated genes;(2)Go and KEGG pathway analysis showed that the down regulated genes of differential expression in PTC significantly were enriched in 18 signaling pathways,and the up regulated genes of differential expression significantly were enriched in 83 signaling pathways.Meanwhile,many signaling pathways have been confirmed to be closely related to tumor development;(3)The results of RT-qPCR showed that the mRNA expression of 5 up-regulated genes and 5 down-regulated genes was significantly different between PTC tumor tissue and adjacent noncancerous samples,and the difference was statistically significant,which was consistent with RNA-seq results.The results of public database analysis showed that the expression of SLC34A2、SFTPB、TACSTD2、FN1、CHI3L1、MT1G、TPO、MT1Hand TFF3 in PTC cancer tissues were significantly different from those in normal thyroid tissues except PKD1L1.(4)The most significant module and key gene FN1 was screened out by constructing PPI network.(5)The expression of FN1 was up-regulated in human papillary thyroid carcinoma cell line,FN1 expression level was closely related to age and N stag.The correlation analysis results of 52 PTC patients collected in this study with clinicopathological parameters showed that their expression levels were significantly different in PTC patients with different N stages(p=0.004).There were no significant differences in stages.Spearman correlation analysis results showed that FN1 expression level was significantly positively correlated with N stage(p=0.004,rs=0.5),and had no significant correlation with age,gender,T stage,TNM stage.The ROC curve AUC value of FN1 is 0.916.It has been proved from public databases that FN1 is over-expressed in PTC,and its expression level was higher in thyroid cancer cell lines and many kinds of tumors,especially in papillary thyroid cancer.TCGA data analysis showed that the abnormal expression of FN1 was positively correlated with T stage,N stage and TNM stage(AJCC)of PTC,negatively correlated with DNA methylation,and not significantly correlated with age,gender and M stage.The higher the expression level of FN1,the worse the disease-free survival of PTC patients(P=0.026).According to cbioportal data,4%of PTC patients have FN1 gene mutation,which was mainly manifested in mRNA up regulation.Conclusion:This study explored the differential expression of the whole gene transcriptome level of papillary thyroid cancer in northeastern Sichuan,and obtained a large number of potential candidate genes closely related to the incidence of PTC;itmay play an important role in the occurrence and development of PTC,and can be used as an important molecular marker for early diagnosis and prognostic evaluation of PTC,and provides new ideas for molecular targeted therapy of PTC.