Background: The molecular mechanisms of EWS-FLI-mediating target genes and downstream pathways may provide a new way in the targeted
therapy of
Ewing sarcoma. Meanwhile, enhancers transcript non-coding RNAs, known as
enhancer RNAs (
eRNAs), which may serve as potential diagnosis markers and therapeutic targets in
Ewing sarcoma. Materials and methods: Differentially expressed genes (DEGs) were identified between 85
Ewing sarcoma samples downloaded from the Treehouse database and 3 normal bone samples downloaded from the Sequence Read Archive database. Included in DEGs, differentially expressed
eRNAs (DEeRNAs) and target genes corresponding to DEeRNAs (DETGs), as well as the differentially expressed TFs, were annotated. Then, cell type identification by estimating relative subsets of known
RNA transcripts (CIBERSORT) was used to infer portions of infiltrating immune cells in
Ewing sarcoma and normal bone samples. To evaluate the prognostic value of DEeRNAs and immune function, cross validation, independent prognosis analysis, and Kaplan-Meier survival analysis were implemented using
sarcoma samples from the
Cancer Genome Atlas database. Next, hallmarks of
cancer by gene set variation analysis (GSVA) and immune gene sets by single-sample gene set enrichment analysis (ssGSEA) were identified to be significantly associated with
Ewing sarcoma. After screening by co-expression analysis, most significant DEeRNAs, DETGs and DETFs, immune cells, immune gene sets, and hallmarks of
cancer were merged to construct a co-expression regulatory network to eventually identify the key DEeRNAs in
tumorigenesis of
Ewing sarcoma. Moreover, Connectivity Map Analysis was utilized to identify small molecules targeting
Ewing sarcoma. External validation based on multidimensional online databases and
scRNA-seq analysis were used to verify our key findings. Results: A six-different-dimension regulatory network was constructed based on 17 DEeRNAs, 29 DETFs, 9 DETGs, 5 immune cells, 24 immune gene sets, and 8 hallmarks of
cancer. Four key DEeRNAs (CCR1, CD3D, PHLDA1, and RASD1) showed significant co-expression relationships in the network. Connectivity Map Analysis screened two candidate compounds,
MS-275 and
pyrvinium, that might target
Ewing sarcoma. PHLDA1 (key DEeRNA) was extensively expressed in cancer stem cells of
Ewing sarcoma, which might play a critical role in the
tumorigenesis of
Ewing sarcoma. Conclusion: PHLDA1 is a key regulator in the
tumorigenesis and progression of
Ewing sarcoma. PHLDA1 is directly repressed by EWS/FLI1
protein and low expression of FOSL2, resulting in the deregulation of FOX
proteins and
CC chemokine receptors. The decrease of infiltrating T-lymphocytes and TNFA signaling may promote
tumorigenesis and progression of
Ewing sarcoma.