Ewing's sarcoma（ES）是儿童和青少年常见的骨恶性肿瘤，严重影响患者的预后。本研究的目的是确定ES的新生物标记和潜在治疗靶点。通过独立预后分析鉴定了高度预后相关的中心基因，并在GSE17679数据集上进行了生存分析，Cox回归分析和临床相关分析。然后，我们在GSE63157，GSE45544和GSE73166数据集上验证了这些关键基因。根据关键基因的高低表达筛选不同表达基因（DEG），并执行基因本体（GO），Kyoto基因和基因组百科全书（KEGG）通路分析和基因集富集分析（GSEA），以及基因集变化分析（GSVA）以探索ES的潜在机制，并根据蛋白质-蛋白质相互作用（PPI）网络建立了重要的模块基因。此外，分析了模块基因和免疫微环境之间的相关性，并使用TIMER和TISIDB研究了关键基因和肉瘤免疫浸润水平之间的相关性。最后，通过实时定量PCR（RT-qPCR），对ES细胞系（RD-ES和A673细胞）中这些关键基因的表达水平进行了进一步验证。使用CCK-8和EdU实验评估ANXA1敲低对RD-ES细胞增殖的影响。鉴定了ANXA1作为ES预后的关键基因。低ANXA1表达的患者的总生存时间较短，并且转移组中ANXA1的表达水平显著低于原发组（P＜0.01）。此外，ANXA1低表达组中12种免疫细胞的丰度显著低于高表达组（所有P＜0.05），这可能与免疫微环境的抑制有关。根据96个DEG构建了PPI网络，进一步鉴定了与ANXA1相关的五个模块基因（COL1A2，MMP9，VIM，S100A11和S100A4）。在验证两个ES细胞系后，ANXA1，COL1A2，MMP9，VIM，S100A11和S100A4的表达水平在ES细胞系和间充质干细胞之间显著不同（所有P＜0.01）。在这些基因中，ANXA1，COL1A2，MMP9，VIM和S100A4与ES患者的预后显著相关（都P＜0.05）。重要的是，ANXA1敲低显著促进了RD-ES细胞的增殖，这可能解释了ANXA1低表达组易于ES转移的原因。ANXA1可能作为ES患者的独立预后生物标志物，并与ES的转移和免疫抑制微环境相关，需要在进一步研究中进行验证。

Ewing's sarcoma (ES) is a common bone malignancy in children and adolescents that severely affects the prognosis of patients. The aim of this study was to identify novel biomarkers and potential therapeutic targets for ES.Highly prognosis-related hub genes were identified by independent prognostic analysis in the GSE17679 dataset. We then performed survival analysis, Cox regression analysis and clinical correlation analysis on the key gene and validated them with the GSE63157, GSE45544 and GSE73166 datasets. Differentially expressed genes (DEGs) were screened based on the high and low expression of key gene, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, Gene Set Enrichment Analysis (GSEA), and Gene Set Variation Analysis (GSVA) were performed to explore the underlying mechanisms of ES, and significant module genes were established based on protein-protein interaction (PPI) networks. Furthermore, the correlations between module genes and the immune microenvironment were analyzed and the correlations between the key gene and immune infiltration levels in sarcoma were investigated using TIMER and TISIDB. Finally, the expression levels of these key genes in ES cell lines (RD-ES and A673 cells) were further validated by real-time quantitative PCR (RT-qPCR). CCK-8 and EdU assays were performed to assess the effect of ANXA1 knockdown on RD-ES cell proliferation.ANXA1 was identified as a key gene for ES prognosis. The overall survival (OS) time of patients with low ANXA1 expression was shorter, and the expression level of ANXA1 in the metastatic group was significantly lower than that in the primary group (P<0.01). Additionally, the abundance of 12 immune cells in the ANXA1 low-expression group was significantly lower than that in the high-expression group (all P<0.05), which may be related to the inhibition of the immune microenvironment. A PPI network was constructed based on 96 DEGs to further identify the five ANXA1-related module genes (COL1A2, MMP9, VIM, S100A11 and S100A4). The expression levels of ANXA1, COL1A2, MMP9, VIM, S100A11 and S100A4 were significantly different between ES cell lines and mesenchymal stem cells after validation in two ES cell lines (all P<0.01). Among these genes, ANXA1, COL1A2, MMP9, VIM and S100A4 were significantly associated with the prognosis of ES patients (all P<0.05). Importantly, ANXA1 knockdown significantly promoted the proliferation of RD-ES cells, which may explain the susceptibility to ES metastasis in the ANXA1 low-expression group.ANXA1 may serve as an independent prognostic biomarker for ES patients and is associated with metastasis and the immunosuppressive microenvironment in ES, which needs to be validated in further studies.