| Backgroud:The thyroid gland is the largest endocrine organ in the human body,which maintains normal physiological function by continuously producing and secreting thyroid hormones T3 and T4.After the thyroid hormones enter the bloodstream,they are transported to various organs throughout the body.The incidence of thyroid carcinoma(THCA)are increasing year by year.According to statistics from the National Cancer Center of China,the incidence of thyroid cancer in China was1.78/100,000 from 1988 to 1992,and it has risen to 12.40/100,000 in 2014.The latest global cancer incidence data for 2018 shows that the standardized incidence rate of thyroid cancer in China is approximately 10.1/100,000,with incidence rates for men and women at 4.8/100,000 and 15.8/100,000,respectively.The number of female cases is about 3.3 times that of male cases.Thyroid cancer has become one of the most common cancers with the highest incidence rate among urban women.Worldwide,the incidence rate of thyroid cancer is increasing at a rate of 6%per year,making it the fastest growing malignant tumor.Differentiated thyroid cancer patients usually undergo thyroidectomy,radioactive iodine therapy for high-risk recurrence patients,and postoperative routine suppression therapy with thyroid-stimulating hormone,and most of them have excellent long-term prognosis.However,a small percentage of thyroid cancer(especially anaplastic thyroid cancer)has a poor prognosis and is highly invasive.The one-year survival rate is only 10%to 20%,and the mortality rate is over 90%.Glycoproteins are important signaling molecules in the human body.Among them,sialic acid-binding immunoglobulin-like lectins(Siglecs)can recognize glycan structures containing sialic acid,mediate cell-to-cell or cell-to-pathogen interactions,and play an important regulatory role in innate and adaptive immunity.Recent studies have shown that one of the family member of Siglecs,Siglecs-15,can continuously inhibit T cell activity and has the main characteristics of immune normalization therapy,but there are fewer studies on Siglecs-15 in thyroid cancer.Therefore,this study aims to explore the biological relationship between Siglecs-15 and thyroid cancer and provide theoretical support for exploring the application of Siglecs-15 in the treatment of refractory thyroid cancer.Objective:To investigate the immune correlation of Siglecs-15 in thyroid cancer and its correlation with clinicopathological features,and to further clarify its biological function in thyroid cancer.Methods:1.Via deep analysis of data from TCGA and IMMPORT database,we screened for immune-related genes involved in immune regulation and differentially expressed in the THCA.Bioinformatics methods were used to further analyze the gene annotation-related signaling pathway hub genes of these differentially expressed immune-related genes and their correlation with survival prognosis.2.Bioinformatics methods were used to further analyze the difference between the groups with high and low Siglec-15 expression,and the protein-protein interaction network was constructed using STRING database,and the effects of Sigrec-15 on infiltrating immune cells in thyroid cancer and the differences in immune-infiltration scores were verified and analyzed.3.Tissue microarrays were constructed,and immunohistochemistry was used to detect the expression of Siglecs-15 in 110 thyroid cancer tissue specimens and 54adjacent tissue specimens.Clinical data were collected,and the correlation between Siglecs-15 expression and clinical pathological features was analyzed.4.Western blot was used to detect Siglecs-15 expression in thyroid cancer cell lines.Siglecs-15 Si RNA antibody was used to knockdown Siglecs-15 expression in ARO and FRO thyroid cancer cell lines.q PCR and Western blot were used to detect cell transfection efficiency and knockdown effect.Colony formation assay was used to detect cell cloning ability,MTT assay was used to detect cell proliferation ability,flow cytometry was used to detect cell apoptosis,and Transwell assay was used to detect cell migration and invasion ability.Co-IPWB was used to analyze the mechanism of Siglecs-15 regulation of STAT3.Subsequently,recovery experiments were designed to test whether Siglecs-15 promotes THCA cell proliferation and migration by activating STAT3.5.A nude mouse xenograft model was established to verify the regulatory role of Siglecs-15 in thyroid cancer.Results:1.Based on the THCA expression profile from TCGA database,we obtained 3422DEGs(1748 upregulated genes and 1674 downregulated genes).We downloaded 2260immune-related genes from the IMMPORT database.Through Venn analysis,there were 362 common genes between these two datasets,which were identified as THCA-related immune genes.GO analysis showed that these common genes were mainly enriched in GO terms such as cell chemotaxis,T cell receptor complex,and signal receptor activator activity.KEGG analysis revealed that these 362 common genes were significantly associated with primary immunodeficiency,JAK-STAT signaling pathway,PD-L1 expression,and PD-1 checkpoint pathway.2.Via the meturquoise module we found 275 immune genes related to thyroid,which was seen as the hub genes.We performed single-factor Cox regression analysis on the 275 immune-related hub genes and found that 23 genes were significantly associated with the overall survival(OS)of thyroid cancer.Multi-COX analysis revealed that 15 of these genes could serve as independent prognostic genes for thyroid cancer.The risk score distribution curve showed that high-risk THCA patients had a shorter survival time than low-risk patients.The ROC curve confirmed that the prognostic markers had high specificity and sensitivity.The AUC value of the Siglecs-15-ROC curve was the highest,at 0.891.3.Compared to normal tissue,Siglecs-15 m RNA expression was upregulated in thyroid cancer tissue.Compared to paired adjacent tissue,Siglecs-15 m RNA expression was still upregulated,and this result was validated by GEO dataset.In clinical samples,the expression level of Siglecs-15 was significantly increased in thyroid cancer tissue compared to adjacent normal tissue(p<0.001).Gene set enrichment analysis(GSEA)results showed that high expression of Siglecs-15 m RNA was positively correlated with many immune pathways.The examination results of the main features of immunology show that high expression of Siglecs-15 m RNA is mainly positively correlated with a large number of cancer immune modulators and pathways.And the upregulation of Siglecs-15 is positively correlated with enhanced immune scores,matrix scores,and estimated scores.4.The Siglecs-15 gene does not show any significant mutations in thyroid cancer.However,in patients with high expression of Siglecs-15,most of the mutations are located in the BRAF gene,while patients with low expression of Siglecs-15 have mutations in the BRAF,NRAS,and HRAS genes.High expression of Siglecs-15 in clinical samples of thyroid cancer is positively correlated with lymph node metastasis and capsule invasion(p=0.035,p=0.002)in patients.Additionally,the expression of Siglecs-15 is higher in lateral cervical lymph node metastasis than in central lymph node metastasis(p=0.009).5.After treatment with agonistic anti-CD40 antibody(5C11)and Siglecs-15 RNA interference,inhibited THCA cell proliferation,promoted apoptosis,and inhibited THCA cell migration and invasion.Siglec-15 silencing was superior to agonistic anti-CD40 monoclonal antibody(5C11)in inhibiting cell proliferation and promoting apoptosis.Furthermore,the inhibitory effect of Siglecs-15 silencing was superior to those of agonistic anti-CD40 antibody(5C11)treatment.Western blot results showed that silencing Siglecs-15 expression in thyroid cancer cells could significantly inhibit the expression of STAT3 and VEGF,and increase the expression of caspase-3.Co-IP results show that Siglecs-15 and STAT3 interact with each other.After silencing Siglecs-15,the expression of STAT3 protein decreased.However,after IL-6 stimulation,the expression of STAT3 increased.Siglecs-15 promotes the proliferation,migration,and invasion of thyroid cancer cells,and inhibits apoptosis by regulating the STAT3signaling pathway.Functional recovery experiments show that Siglecs-15 promotes the malignant phenotype of thyroid cancer cells by activating the STAT3 signaling pathway.Mouse models were constructed with Siglecs-15 RNAi and overexpression of STAT3+Siglecs-15 RNAi.6.In thyroid carcinoma mouse models,Silencing Siglec-15 significantly inhibited the growth rate and tumor volume of thyroid carcinoma cell xenografts,inhibited Ki67and VEGF protein expression,and increased the CD4~+/CD8~+ratio And it can reverse the regulation of Siglec-15 through STAT3.Conclusion:1.Siglecs-15 is an immune-related hub gene in the THCA dataset and has potential diagnostic value for thyroid cancer.2.Siglecs-15 plays an important role in thyroid cancer and is associated with the immune microenvironment of thyroid cancer,making it a promising immune checkpoint for thyroid cancer.3.Silencing of Siglecs-15 expression can inhibited in the proliferation,migration and invasion of thyroid cancer cells.4.Siglec-15 affects the proliferation,migration,and invasion of thyroid cancer cells by activating the STAT3 signaling pathway,and participates in regulating immune function. |
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