| Background and Objectives:Ewing Sarcoma(ES)is a malignant bone and soft tissue tumor which was thought to arise from mesenchymal stem cells.It is the second most common osteogenic tumor in pediatric patients,with an incidence of about 1 case per 1.5 million people.ES can occur anywhere in the body,with 80% occurring in bone,most commonly in the pelvis and proximal to long bones.Extraosseous ES is more common in adults and mainly occurs in paravertebral and thoracic soft tissues.Current management of primary ES,which relies on a combination of cytotoxic drugs and local reduction through surgery,radiotherapy,or both,according to feasibility,has improved the 5-year survival rate among patients with localized disease to about 70% currently.However,nearly 25% of patients were found metastasis when diagnosed.The 5-year OS of them is less than 30%,and conventional treatment is difficult to achieve ideal results in metastatic patients.Exploring the underlying molecular mechanisms of Ewing’s sarcoma and identifying effective targeted therapies and prognostic biomarkers will provide more opportunities to overcome current clinical difficulties.The microenvironment that tumor cells depend on for growth and survival plays an important role in the occurrence and progression of tumor.The tumor part is composed of a complex tumor microenvironment(TME),which includes not only tumor cells but also resident fibroblasts(cancer-associated fibroblasts;CAFs)and macrophages,distant recruited infiltrating immune cells such as myeloid cells and lymphocytes,bone marrowderived cells(BMDCs)such as endothelial progenitor cells and hematopoietic progenitor cells,etc.These cells can secrete various cytokines,chemokines,growth factors and new blood vessels to regulate the occurrence and development of tumors.Therefore,by analyzing the heterogeneity and complexity of tumor microenvironment,it is possible to identify the immune characteristics of different tumors,improve the ability to guide and predict the immunotherapy response,and facilitate the search for new therapeutic targets.This study intends to analyze and evaluate the immune characteristics of ES from the perspective of TME,explore the genes and potential mechanisms related to tumor microenvironment that regulate the occurrence and progression of ES.Chapter 1 Prognostic analysis and gene screening based on the characteristics of tumor microenvironment in Ewing sarcomaMethods: After integrating ES m RNA chip datasets GSE63155/GSE63156,ESTIMATE analysis is used to calculate tumor purity,stromal cell and immune cell infiltration of ES patientss.WGCNA algorithm was then used to explore the gene modules associated with the tumor microenvironment of ES(ES-TME).We performed univariate Cox survival analysis and Lasso regression analysis on the genes of the ES-TME genesets,screened the prognostic candidate genes and constructed a prognostic model(ES Score)to evaluate their clinical evaluation effect on ES.We used sequencing data of ES from the ICGC database to test the efficacy of the prognostic model and the reliability of the candidate genes to identify the key TME gene that is critical for prognosis.Finally,we detected the expression of this key TME gene in various types of cancers based on TCGA database to evaluated its role in malignant tumors preliminatively,and analyzed its potential immunomodulatory function.Result: Through bioinformatics analysis,we found that both immune score and stromal score calculated by the ESTIMATE algorithm were prognostic risk factors and were significantly negatively associated with tumor purity.Further screening based on immune microenvironment score showed that ARAP3,PODXL,CCL18 and CTSC were TME candidate genes associated with prognosis of ES,and a prognostic model was then established.Model evaluation and Survival analysis suggested that ARAP3 was a key adverse prognostic factor affecting OS and EFS in both GEO and ICGC databases.Combined with literature learning,we finally concluded that ARAP3 may be the candidate key ES-TME regulator of ES.TCGA expression profile analysis indicated that ARAP3 expression was quite different between tumor types,and was significantly low in breast cancer,lung cancer,cervical cancer and other tumors.It is highly expressed in cholangiocarcinoma,prostate cancer,pancreatic cancer and other tumors,suggesting that the role and phenotype of ARAP3 in different tumors have great heterogeneity.Further analysis indicated that ARAP3 was involved in many immune activities and might be a potential immunomodulatory target.Conclusions: The immune microenvironment of ES plays an important role in regulating the progression of ES.Through the immune score,stromal score and related prognostic analyses of ES datasets,it was found that ARAP3 could be a potential target of ES.Chapter 2 Role of ARAP3 in malignant biological behavior of Ewing sarcomaMethods: The expression profile of ARAP3 was determined by qRT-PCR,Western Blot and IHC staining in ES cell lines and tissue samples from clinical patients.ARAP3 was then knocked down in ES cell lines for further analysis.The regulation of ARAP3 on malignant biological behavior of ES was investigated in vitro by clonal formation assay,CCK8 assay,Transwell assay,apoptosis assay and cell cycle assay.Finally,total m RNA of si-NC and si-ARAP3 ES cells was collected for RNA-seq,and pathway analysis was performed to explore the potential mechanism of ARAP3 in regulating malignant biological behavior of ES.Result: The expression of ARAP3 in ES was significantly higher than that in adjacent normal tissues.The expression of ARAP3 was significantly higher in ES cell lines than in normal h BMSC.We designed si RNA to knock down the expression of ARAP3 in RDES and SK-ES-1 cells.It was found in vitro that knocking down ARAP3 significantly inhibited the proliferation of tumor cells and induced apoptosis of tumor cells,as well as the migration and invasion ability.RNA-seq and PCR confirmed the activation of p53 signaling pathway after ARAP3 knockdown.Conclusions: ARAP3 was highly expressed in ES.Knockdown of ARAP3 can activate the p53 signaling pathway to inhibit the proliferation,migration and invasion and promote the apoptosis of ES cells.Chapter 3 Regulation and potential mechanism of ARAP3 on microenvironmentMethods: We performed CIBERSORT and ss GSEA algorithm on ES data from GEO and ICGC databases to evaluate the proportion of immune cells in ES microenvironment and the differences in immune cell infiltration between ES patients.The correlation between ARAP3 and macrophage infiltration in ES was further analyzed based on the algorithm results and IHC staining of clinical samples.We used Monocyte recruitment assay to determine the effect of ARAP3 on chemotaxis of BMM and RAW264.7 cells.CM of siNC and si-ARAP3 ES cells were collected.RAW264.7 cells were induced into osteoclast,with CM added into the induced system,to investigate the effects of ARAP3 on osteoclasts differentiation.Finally,RNA-seq was used to explore the potential mechanism of ARAP3 affecting the microenvironment modification of ES.Result: CIBERSORT analysis showed that macrophages were abundant among infiltrated immune cells of ES patients,and M2 type macrophages accounted for a large proportion.ss GSEA analysis showed that ARAP3 expression was significantly positively correlated with macrophage infiltration.IHC staining on tissue chip further demonstrated that ARAP3 expression was positively correlated with macrophage marker gene CD68 expression in ES samples.In vitro experiments confirmed that knocking down of ARAP3 inhibited the recruitment effect of ES cells on BMM and RAW264.7 cells.Through osteoclast differentiation assays,we found that CM of si-ARAP3 ES cells could inhibit osteoclast differentiation.RNA-seq、q RT-PCR and WB showed that the expression of IL1 B was significantly decreased after ARAP3 knockdown,which may be the key factor of the role of ARAP3 in the microenvironment.Conclusions: ARAP3 may affect macrophage recruitment and osteoclast differentiation by regulating secretion of IL1 B. |
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