| Background:Thyroid cancer is the most common malignant tumor in the endocrine system.The pathological types of thyroid cancer include differentiated thyroid cancer（DTC）,anaplastic or undifferentiated thyroid cancer（ATC）,and medullary thyroid cancer（MTC）.Among them,differentiated thyroid cancer includes papillary thyroid carcinoma（PTC）,follicular thyroid cancer（FTC）,and poorly differentiated thyroid cancer（PDTC）.Among these five types,PTC is the most common,accounting for nearly 80%of thyroid cancers.Although PTC patients are usually well-differentiated thyroid cancer and have a relatively good prognosis,about 20%of PTC patients may experience recurrence and metastasis after surgery.Even worse,some recurrent PTC may transform into poorly differentiated or undifferentiated thyroid cancer with a very poor prognosis.Lymph node metastases（LNM）are important indicators of PTC recurrence and distant metastasis.The rate of lymph node metastasis in PTC is as high as 84.7%,and lymph node metastasis increases the probability of postoperative recurrence and mortality.Therefore,most surgeons currently perform prophylactic lymph node dissection（ND）on PTC patients.However,studies have found that ND does not improve the survival rate of PTC patients,which is related to the increase in surgical complications.Worse still,about 54.1%of lymph node metastasis patients.have occult central neck lymph node metastasis,and these patients may require a second surgery,which means more and more serious postoperative complications.Therefore,on one hand,it is urgently necessary in clinical practice to predict the lymph node metastasis of PTC patients using biomarkers,which can guide doctors to perform lymph node dissection more accurately,provide a basis for reducing postoperative complications,and improve survival rate and quality of life.On the other hand,it is also imperative to explore the mechanism and targets of PTC lymph node metastasis,and to take effective measures or targeted therapy based on accurate prediction of lymph node metastasis,which can inhibit the lymph node metastasis and recurrence of PTC and improve the survival quality of PTC patients.The application of bioinformatics methods in the field of medicine can integrate multi-center and multi-omics data,comprehensively screen disease targets,explore the potential mechanisms of diseases,and construct prognostic models.By combining basic experiments and clinical research,the diagnostic efficacy of prognostic markers can be analyzed,and more effective targeted drugs can be developed,providing a theoretical basis for personalized and precise treatment of clinical diseases.In this study,bioinformatics methods were applied to analyze the correlation between differential genes and clinical features by integrating multiple databases,and TM4SF1 was identified as a potential driver gene for PTC lymph node metastasis.TM4SF1（Transmembrane-4-L-Six-Family-1）,also known as tumor-associated antigen L6,is a member of the four-transmembrane 4-L6 family and is a 22 kDa protein with four transmembrane domains.Studies have shown that TM4SF1 not only promotes tumor progression and lymphangiogenesis by regulating lymphatic vessel formation markers in various malignant tumors such as pancreatic cancer,colon cancer,rectal cancer,and liver cancer but also is closely related to the clinical pathological staging of various tumors.This suggests that the role of TM4SF1 in tumor lymph node metastasis should not be ignored,but the relationship between TM4SF1 and PTC lymph node metastasis still needs to be explored.In summary,this study applied bioinformatics methods,PTC tissues,PTC cells,and a lymph node metastasis animal model to study the expression of TM4SF1 in PTC and its relationship with clinical pathology.The study explored the biological function of TM4SF1 in PTC and investigated the mechanism of its role in PTC lymph node metastasis.This provides new clues and ideas for TM4SF1 as a molecular marker and therapeutic target in PTC.Chapter 1:Expression of TM4SF1 in PTC and its correlation with clinical pathological featuresResearch objectives:Analyze the expression of TM4SF1 in the PTC lymph node metastasis group（N1）and the lymph node non-metastasis group（N0）,and validate it in clinical tissue samples;analyze the relationship between the expression level of TM4SF1 and a number of clinical pathological features;construct a lymph node metastasis regression model to analyze the diagnostic efficacy of TM4SF1 as a molecular marker for PTC lymph node metastasis;and preliminarily explore the mechanism of high expression of TM4SF1 in the PTC N1 group.Research methods:1.Data mining of public databases TCGA and GEO was conducted to analyze the expression of TM4SF1 in PTC N1 and NO groups,and its relationship with clinical pathological features.A binary logistic regression analysis method was used to establish a lymph node metastasis model,and ROC curve analysis was performed to evaluate the diagnostic efficacy of TM4SF1 as an analytical marker for predicting PTC lymph node metastasis.2.qRT-PCR and immunohistochemistry were used to detect TM4SF1 mRNA and protein levels in Nl and NO PTC tumor tissues.3.Methylation analysis was performed on PTC patient data in the TCGA database to explore the relationship between TM4SF1 methylation levels at various sites and TM4SF1 mRNA expression.4.MSPCR was used to detect TM4SF1 DNA methylation levels in N1 and NO PTC tumor tissues.Research results:1.In the GEO database,there were 127 differentially expressed genes in GSE60542 and 87 differentially expressed genes in GSE129562,grouped by lymph node metastasis.The intersection genes were TM4SF1,SLPI,and RASD1.2.The expression of TM4SF1,SLPI,and RASDl in PTC was validated in the TCGA database,and the results showed that only TM4SF1 had a significant difference between the PTC N1 and NO groups（N1>N0,P<0.001）.3.qRT-PCR and IHC showed that the TM4SF1 mRNA level（P<0.001）and protein level（P<0.01）in PTC N1 tumor tissue were both higher than those in NO tumor tissue.4.The expression level of TM4SF1 was higher in both Nla and Nlb than in the NO group（P<0.01）,and the survival period of both N1a and N1b was lower than that of the NO group（P<0.05）.5.TM4SF1 expression showed statistical differences in different T staging,N staging,and Stage grouping.TM4SF1 mRNA expression was higher in T3-T4 stage（P<0.05）,N1 stage（P<0.0001）,and Stage Ⅲ-Ⅳ（P<0.001）.There was no significant difference in TM4SF1 expression in different age,gender,and M staging（P>0.05）.6.The proportion of lymph node metastasis in PTC was higher in males and patients under 45 years old（P<0.05）.7.TM4SF1,age<45 years,and gender（male）were independent risk factors for lymph node metastasis in PTC.The predicted regression model was Y=-1.621+0.504TM4SF1 expression level-0.494gender-0.014*age,with an ROC curve area of 0.714.8.A negative correlation was found between the mRNA expression level of TM4SF1 and methylation levels of cg00244111（R=-0.106）,cg06800962（R=0.701）,cg08124030（R=-0.536）,cg09442403（R=-0.284）,cg16705300（R=0.106）,cg16810293（R=-0.63）,cg18461436（R=-0.594）,and cg23246821（R=0.71）（all P<0.05）.The methylation levels of cg02857726,cg08124030,cg16705300,and cg18461436 were independent influencing factors for TM4SF1 mRNA expression.9.Methylation analysis of PTC in the TCGA database showed that the methylation levels of TM4SF1 at cg06800962,cg08124030,cg09442403,cg16705300,cg16810293,cg18461436,and cg23246821 sites were lower in the N1 group than in the NO group（P<0.05）.MSPCR detection of methylation in tumor tissues of PTC patients in the N1 and NO groups showed that There was a marked difference between NO and N1 in methylation rates（16.7%versus 66.74%）（P<0.05）.Conclusion:1.Bioinformatics analysis and clinical sample validation（qRT-PCR and immunohistochemistry）suggest that TM4SF1 is highly expressed in PTC with lymph node metastasis.2.TM4SF1 mRNA expression is higher in PTC patients with T3-T4 stage,N1 stage,and Stage Ⅲ-Ⅳ,while no significant differences were observed in different ages,genders,and M stages.TM4SF1 high expression,male gender,and age≤45 years are associated with higher lymph node metastasis rates in PTC patients.TM4SF1,age,and gender are independent risk factors for PTC lymph node metastasis.The combination of the three factors has a certain diagnostic value in predicting lymph node metastasis.3.There is a negative correlation between TM4SF1 expression and DNA promoter methylation.TM4SF1 is less methylated in PTC N1 group.Chapter 2:High expression of TM4SF1 promotes epithelial-mesenchymal transition and lymph node metastasis in PTCTo explore the biological function of TM4SF1 in PTC through in vitro and in vivo experiments,and to investigate the effects of TM4SF1 silencing and overexpression on epithelial-mesenchymal transition（EMT）and migration of PTC cells,and the impact of TM4SF1 silencing on subcutaneous tumorigenesis and lymph node metastasis in nude mice.Research methods:1.Determine the optimal MOI and infection time of lentivirus-mediated TM4SF1 silencing and overexpression in PTC cells.2.Infect PTC cells with lentivirus-mediated TM4SF1 silencing and overexpression under the optimal conditions,screen stable transfected cells and culture them in passage.3.Observe the effect of TM4SF1 silencing and overexpression on PTC cell proliferation and migration ability through colony formation and Transwell assays.4.Detect the expression levels of EMT markers in PTC cells after TM4SF1 silencing and overexpression by Western blot.5.Inject TM4SF1-silenced and control PTC cells subcutaneously into the footpads of nude mice to establish a popliteal lymph node metastasis model.Measure the weight of the transplanted tumors and popliteal lymph nodes,and prepare pathological sections of the transplanted tumors and lymph nodes.Observe cell morphology by HE staining and detect the expression levels of LYVE1 by immunohistochemistry.Research results:6.The optimal conditions for lentivirus-mediated TM4SF1 silencing and overexpression in PTC cells were MOI=50 and complete culture medium with HitransG P for 72 hours.7.The efficiency of TM4SF1 silencing and overexpression was over 95%for both lentivirus-mediated groups as observed under fluorescence microscopy and bright-field microscopy.In the silencing group,TM4SF1 protein expression was significantly lower than in the control group（P<0.05）,while the TM4SF1 overexpression group showed significantly higher expression of TM4SF1 protein than the control group（P<0.05）.8.The colony formation assay showed that TM4SF1 overexpression significantly increased PTC cell proliferation and colony formation ability（P<0.01）,while TM4SF1 silencing significantly reduced PTC cell proliferation and colony formation ability（P<0.01）.The Transwell assay showed that TM4SF1 overexpression significantly increased PTC cell migration ability（P<0.001）,while TM4SF1 silencing significantly reduced PTC cell migration ability（P<0.01）.9.According to Western blot results,compared to the control group,TM4SF1 overexpression significantly reduced the expression levels of the epithelial cell marker E-cadherin（P<0.01）and TIMP1（P<0.01）,and significantly increased the expression level of the mesenchymal cell marker N-cadherin（P<0.05）.TM4SF1 silencing significantly increased the expression levels of E-cadherin（P<0.01）and TIMP1（P<0.01）,and significantly reduced the expression levels of N-cadherin（P<0.05）,SNAI1（P<0.01）and MMP9（P<0.01）.10.Transplanted tumors and popliteal lymph nodes in the TM4SF1 silencing group weighed significantly less than those in the control group（P<0.01 and P<0.05,respectively）.The expression level of LYVE1 in the poplitealConclusion:1.Overexpression of TM4SF1 can promote proliferation,colony formation,and migration of PTC cells,while silencing TM4SF1 can inhibit these processes.2.Overexpression of TM4SF1 can promote epithelial-mesenchymal transition of PTC cells,while silencing TM4SF1 can inhibit this process.3.In the nude mouse subcutaneous tumor and popliteal lymph node metastasis model,silencing TM4SF1 can inhibit tumor growth,lymphatic vessel formation,and lymph node metastasis of PTC.Chapter 3:Mechanism study of TM4SF1 regulating the growth and lymph node metastasis of PTCResearch objective:To explore the potential mechanisms of the TM4SF1 gene in papillary thyroid carcinoma（PTC）and further verify that the TM4SF1 gene may promote PTC progression and lymph node metastasis by regulating the Wnt/β-catenin signaling pathway and immune cell infiltration.Research methods:1.TCGA database was used to obtain gene expression data of PTC patients,and GSEA enrichment analysis was performed to explore the correlation between the TM4SF1 gene and the Wnt/β-catenin signaling pathway.2.WB was used to detect the expression levels of Wnt/β-catenin signaling pathway-related proteins in PTC cells with TM4SF1 overexpression or knockdown.3.Immunofluorescence was used to detect β-catenin nuclear translocation in PTC cells with TM4SF1 overexpression or knockdown.4.Immune analysis of PTC patient data in the TCGA database was performed to investigate the relationship between the TM4SF1 gene and immune cell infiltration and lymph node metastasis in PTC.Research results:1.GSEA analysis showed that genes upregulated in the Wnt/β-catenin pathway were enriched in the TM4SF1 high-expression group,and the expression level of TM4SF1 was significantly correlated with the Wnt/β-catenin signaling pathway（P<0.001）.2.There was no significant difference in total β-catenin protein levels between the TM4SF1 overexpression group and control group（P>0.05）,or between the TM4SF1 knockdown group and control group（P>0.05）.3.Compared with the empty vector control group,the cytoplasmic β-catenin protein level was significantly decreased（P<0.01）,and the nuclear β-catenin protein level was significantly increased（P<0.01）in the TM4SF1 overexpression group.In contrast,the cytoplasmic β-catenin protein level was significantly increased（P<0.05）,and the nuclear β-catenin protein level was significantly decreased（P<0.01）in the TM4SF1 knockdown group.4.In comparison with the control group,immunofluorescence results indicated that the nuclear β-catenin fluorescence was significantly enhanced,and the cytoplasmic β-catenin fluorescence was significantly reduced in the TM4SF1 overexpression group,while the nuclearβ-catenin fluorescence was significantly reduced,and the cytoplasmic β-catenin fluorescence was significantly increased in the TM4SF1 knockdown group.5.The levels of GSK3β were not significantly different from the control group in both the TM4SF1 overexpression and knockdown groups,but the level of p-GSK3βwas increased（P<0.05）in the TM4SF1 overexpression group and decreased（P<0.01）in the TM4SF1 knockdown group.6.Compared with the empty vector control group,the protein levels of the Wnt/β-catenin pathway transcription factor TCF1 and the transfer-related target genes MMP7 and MMP9 were significantly increased（P<0.01）in the TM4SF1 overexpression group,while the protein levels of TCF1,MMP7,and MMP9 were significantly decreased（P<0.01）in the TM4SF1 knockdown group.7.The Wnt/β-catenin pathway inhibitor XAV-939 reversed the promotion of PTC cell proliferation,colony formation,and migration caused by TM4SF1 overexpression（P<0.05）,while the Wnt/β-catenin pathway activator SKL2001 reversed the inhibition of PTC cell proliferation,colony formation,and migration caused by TM4SF1 knockdown（P<0.05）.8.In thyroid cancer,the mRNA expression level of TM4SF1 is significantly positively correlated with B cells（R=0.309）,CD4+T cells（R=0.423）,macrophages（R=0.253）,neutrophils（R=0.513）,and dendritic cells（R=0.419）;the infiltration of macrophages,NK cells,and CD4+T（Tregs）cells in the N1 group is significantly higher than that in the NO group（P<0.05）.9.TM4SF1 may regulate Tregs cell infiltration through the IL2/STAT5 signaling pathway to promote lymph node metastasis in PTC.Conclusion:1.TM4SF1 affects the proliferation,colony formation,and migration of PTC cells by regulating the Wnt/β-catenin pathway.2.TM4SF1 enhances GSK3β phosphorylation to dissociate the GSK3β/β-catenin degradation complex,promotes β-catenin nuclear translocation,and ultimately increases the transcription level of transfer-related target genes.3.TM4SF1 may also regulate immune cell infiltration to provide a favorable microenvironment for lymph node metastasis in PTC. |
PDF Full Text Request