The Role Of KLK10 In The Development Of Papillary Thyroid Carcinoma And Its Mechanism

BACKGROUND:Papillary thyroid cancer(PTC)is one of the few tumors with a rapidly increasing incidence that has attracted widespread attention.Recurrence and metastasis can occur in some patients with PTC,but the mechanisms involved are still unclear.In recent years,the development of next-generation sequencing technologies has had a profound impact on the field of tumor genomics.With the massive amount of data generated from tumor samples,The Cancer Genome Atlas(TCGA)database and its affiliated projects were born.Among them,tumor transcriptomics sequencing data has become one of the main tools used to study tumor samples.OBJECTIVES:To screen for the most core differentially expressed genes in PTC and explore their clinical significance.To identify the expression levels of core genes in PTC and explore their molecular mechanisms in PTC recurrence and metastasis.METHODS:1.Download transcriptomic and clinical data of PTC patients from the TCGA database.Deferentially Expressed Genes(DEGs)were obtained and functionally enriched and Protein-Protein Interaction(PPI)networks were constructed.The core genes were obtained by intersecting the top 100 genes in the DEGs and PPIs.2.Independent external sets and other databases were used to validate the expression levels of the CORE GENE in PTC.RT-qPCR and Western Blot were used to further validate the CORE GENE expression in two PTC cell lines and normal thyroid follicular epithelial cell lines as well as the CORE GENE expression levels in thyroid cancer tissues,paracancerous tissues,and normal thyroid tissues of PTC patients.Pan-cancer analysis was also performed to further investigate the pro-carcinogenic effect of the CORE GENE.3.Hazard ratios,diagnostic value,tumor microenvironment scores,immune infiltration abundance,recurrence-free survival curves,and drug sensitivity analysis of clinical indicators in patients with PTC were performed.Stratified analysis of patients by age,gender,and histological type was performed to compare differences in the CORE GENE expression4.Knockdown and overexpression of the CORE GENE in PTC cell lines.CCK-8 was used to detect the effect of the CORE GENE on the proliferative capacity of PTC cells and Transwell assays to detect changes in cell migration and invasive capacity.5.Gene set enrichment analysis of the biological functional changes of PTC upon high the CORE GENE expression to explore the molecular mechanisms.WB was used to detect changes in marker proteins and their phosphorylation levels in the pathways associated with PTC cell lines that knockdown and overexpress the CORE GENE to validate the analysis.RESULTS:1.Transcriptomic data from PTC compared to normal thyroid tissue yielded KLK10 as the only core gene whose expression was upregulated.2.Independent external sets of results all showed that KLK10 expression levels were significantly higher in PTC(p<0.001).RT-qPCR and WB results indicated that KLK10 expression levels were significantly higher in both PTC cell lines than in normal thyroid cell lines.Moreover,KLK10 expression was higher in nail cancer tissues than in paraneoplastic and normal thyroid tissues.3.The results of the logistic regression showed that the risk of lymph node metastasis in PTC was on average 2.29-fold higher when KLK10 expression was elevated,and the risk of tumor progression(T-stage)was also significantly higher(p<0.001).And KLK10 had a high diagnostic value for PTC(AUC of 0.848).The results of the tumor microenvironment score showed that KLK10 expression was positively correlated with all three scores.Furthermore,when KLK10 expression was elevated,most immune cells were significantly more abundantly infiltrated in PTC(P<0.05),relapse-free survival was lower(P=0.034),and PTC was less sensitive to most drugs.Collectively,KLK10 was found to contribute to the clinical progression of PTC in several ways.4.CCK8 assay showed that overexpression of KLK10 enhanced the proliferation of PTC cells(p<0.001),whereas the opposite result was observed when KLK10 was knocked down.transwell assay showed that overexpression of KLK10 significantly promoted the migration and invasion of PTC cells,while knockdown of KLK10 inhibited this phenomenon.5.Data analysis showed that the Akt/mTOR signaling pathway was activated when KLK10 expression was elevated.WB experiments showed that overexpression of KLK10 elevated the phosphorylation levels of Akt and mTOR.CONCLUSION:The findings suggest that elevated KLK10 expression can promote the recurrence and metastasis of PTC and that activation of the Akt/mTOR pathway may be the mechanism responsible for this phenomenon.