Establishment Of A Prognostic Model For Genes Related To Lymph Node Metastasis In Papillary Thyroid Carcinoma And Exploration Of Its Mechanis

Chapter 1:Whole transcriptome sequencing reveal the Long chain RNA landscape of serum exosomes in metastatic papillary thyroid carcinomaBackground:At present,in clinical practice,routine prophylactic lymph node dissection for low-risk PTC patients with negative lymph nodes before operation elevated the rate of postoperative complications,but it is difficult to detect massive occult lymph node metastases with preoperative ultrasound.We aimed to explore reliable markers that can be used to indicate massive occult lymph node metastasis by whole transcriptome sequencing of serum exosomes for clinical decision-making and operation scope.Methods:Serum of patients with PTC was collected before operation.The samples with no lymph node metastasis or with massive lymph node metastasis were screened.Then the serum exosomes were extracted with the qiagen kit,the quality was evaluated by various methods,and the whole transcriptome sequencing was carried out.After comparative analysis,different types of RNAs were analyzed for functional enrichment and pathway analysis.Results:the reliability of exosome extraction was confirmed by WB,TEM and NTA analysis.Sequencing quality was satisfactory and meets the analysis criteria.A total of 974 differential mRNA were obtained,of which 332 were down-regulated and 642 were up-regulated;A total of 1097 differential lncRNAs were obtained,of which 452 were down-regulated and 645 were up-regulated;A total of 29 differential circRNAs were obtained,of which 8 were up-regulated and 21 were down regulated.The functional enrichment of these different RNAs was found to be mainly enriched metabolic regulation,protein synthesis,vesicle fusion,apoptosis regulation,thyroid hormone synthesis and so on.Pathway analysis showed that differential lncRNA target genes and circRNA parent genes were more significantly enriched in tumor related pathways than mRNA.Through the screening of differential RNAs,a total of 10 long-chain RNA with the highest AUC were selected,for further validation on independent samples cohort.Conclusion:the results firstly revealed the whole transcriptome map of serum exosome from patients with metastatic and non-metastatic PTC.The functional enrichment analysis results indicated that compared with mRNA,combination with differential lncRNA and circRNA are more promising in predicting massive occult lymph node metastasis.Exploration and mechanism based on the lymph node metastasis related prognostic model for predicting prognosis of papillary thyroid carcinomaChapter 2:A novel metastasis-related genes-based signature for predicting the progression-free interval of patients with papillary thyroid carcinomaBackground:We aimed to build a novel model with novel epithelial-mesenchymal transition(EMT)signature or metastasis-related genes(MTGs)signature and relevant clinical parameters for predicting progression-free interval(PFI)after surgery for papillary thyroid carcinoma(PTC).Methods:We performed a bioinformatic analysis of integrated PTC datasets with the ERGs or MTGs to identify differentially expressed ERGs or MTGs.Then we generated PFI-related DE-ERGs or DE-MTGs and established signatures.After that,we validated the signatures on multiple datasets.Further,we carried out uni-and multivariate analysis to identify independent prognostic characters.Finally,we established a signature and clinical parameters-based nomogram for predicting the PFI of PTC.Results:We identified 244 DE-ERGs or 155 DE-MTGs related to PFI in PTC.The functional enrichment analysis showed that the DE-ERGs and DE-MTGs were associated with an essential oncogenic process.Consequently,we established novel 10gene signatures and could distinguish patients with poorer prognoses and predicted PFI accurately.Since the efficacy of MTGs signature(C-index of 0.748)was better than ERGs signature(C-index of 0.723),we then optimized the MTGs signature for further analyses.The optimized MTGs signature had a C-index of 0.76 and the relevant nomogram had a C-index of 0.80.Also,it was closely related to pivotal clinical characters of datasets and invasiveness of cell lines.And the signature was confirmed a significant independent prognostic factor in PTC.Finally,we built a nomogram by including the signature and relevant clinical factors.Validation analysis showed that the nomogram’s efficacy was satisfying in predicting PTC’s PFI.Conclusions:The MTG signature and nomogram were closely associated with PTC prognosis and may help clinicians improve the individualized prediction of PFI,especially for high-risk patients after surgery.Chapter 3:Knockdown of SDC2 promote papillary thyroid cancer cells’invasiveness and de-differentiation through enhanced epithelial-mesenchymal transitionBackground:The high incidence of recurrence and metastatic disease remain the major issues for papillary thyroid cancer(PTC)patients.Previous studies have demonstrated that Syndecan-2(SDC2)plays a key role in multiple cancers progression.However,the potential role of SDC2 in PTC progression and recurrence remains unclear.Methods:First,we performed integrated bioinformatics analysis,including immunohistological staining of PTC samples and pairwise normal thyroid samples to explore the potential prognostic value of SDC2.The SDC2 expression-related genes,pathways,and immune infiltration alteration was also identified.Then we applied transient siRNA infection to the knockdown SDC2 expression level in PTC cell line BCPAP.After that,we carried out a scratch wound healing assay and cell counting kits8(CCK8)assay to explore cell migration and viability.Western-blotting on mesenchymal and epithelial markers,RT-qPCR on multiple thyroids differentiating markers,were performed to explore the potential underlying mechanisms.Results:Firstly,we found a significant negative correlation of SDC2 expression with advanced disease characters and found the independent prognostic value in univariate and multivariate analysis.Then bioinformatic analysis indicated the SDC expression was closely related to multiple PTC specifical pathways and immune infiltration environment.Knockdown of SDC2 significantly enhanced the migration ability of BCPAP cells.Further,CCK-8 assay results also indicated the higher cell viability in the condition of SDC2 knockdown.Western blot also revealed the upregulation of α-SMA and the downregulation of ZO-1,indicating the enhanced EMT process.Furthermore,qPCR results also revealed the expression level of thyroid stimulating hormone receptor(THSR)has been reduced,and paired box gene-8(PAX8)elevated.Also,the expression level of components of hedgehog signaling pathway has been reduced,indicating the impact of SDC2 knockdown to the PTC de-differentiation and potential underlying mechanism.Conclusions:Our findings suggest that SDC2 would be a new prognostic marker and a promising target for intervention of advanced thyroid cancer.