| Objective:Ewing’s sarcoma is one of the most common primary bone tumors with high malignancy and poor prognosis,and it mainly occurs in children and adolescents.Currently,a comprehensive treatment plan for Ewing’s sarcoma including local surgical resection,radiation therapy,and chemotherapy is adopted in clinical practice.However,due to its strong metastasis,high recurrence rate,and drug resistance,the prognosis of patients is generally poor,and their quality of life is low.Therefore,the development of new treatment targets and prognostic markers for Ewing’s sarcoma is currently a key issue that urgently needs to be addressed.However,due to the rarity of Ewing’s sarcoma,there is currently a lack of multi-omics data,and the existing transcriptomic data are derived from multiple small sample cohorts,which cannot clearly demonstrate the transcriptomic pathogenic characteristics of this cancer.This paper aims to search for key targets and pathways that may affect the survival of Ewing’s sarcoma patients through the analysis of cDNA microarray and RNA-seq transcriptomic sequencing data from patient samples.Furthermore,through RNAi interference technology combined with cell proliferation,cell cycle,and other experiments,the functional role of the candidate gene CENPF screened in Ewing’s sarcoma is explored.Finally,by combining virtual screening of small molecule inhibitors of related signaling pathways of traditional Chinese medicine natural products with cell-level experiments,the strategy of traditional Chinese medicine natural products for treating Ewing’s sarcoma is preliminarily explored.Methods:This paper’s research is organized into three distinct sections:In the first part,we integrated 10 microarray datasets of Ewing’s sarcoma from the Gene Expression Omnibus(GEO)database and performed normalization to obtain a normalized microarray dataset.Differential gene analysis was conducted to obtain a set of differentially expressed genes between tumor samples and normal samples.Then,we used methods such as gene ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),and gene set enrichment analysis(GSEA)to explore signaling pathways closely related to the occurrence and development of Ewing’s sarcoma.Subsequently,single-gene set grouping prognosis analysis was used to verify the impact of key pathways on prognosis.In addition,immune infiltration analysis was used to obtain a set of differentially infiltrated cells in tumor samples relative to normal samples,and potential genes that may have significant significance for tumor prognosis were identified through survival analysis.Finally,by studying the differential analysis results and survival analysis results,significant biomarker genes in Ewing’s sarcoma were determined,and the inhibitory effects of candidate key genes on the proliferation and colony formation ability of Ewing’s sarcoma cells were studied using RNAi interference technology.In the second part,we conducted pan-cancer analysis of CENPF gene expression,mutation,prognosis,and its correlation with immune checkpoint genes and immune infiltration in various types of cancer using The Cancer Genome Atlas(TCGA)database.Next,we used RNAi interference technology to construct CENPF knockdown cell lines,and studied the effect of CENPF gene on the migration ability of Ewing’s sarcoma cells using scratch assay.We investigated the effect of CENPF gene on the cell cycle of Ewing’s sarcoma cells using flow cytometry technique.Finally,we studied the effect of CENPF gene on proteins related to cell cycle pathway using protein immunoblotting experiment.In the third part,possible genes that may have a significant impact on the progression of Ewing’s sarcoma were screened by integrating GEO microarray datasets and ICGC RNA-seq transcriptomic datasets.The signaling pathways closely related to the occurrence and development of Ewing’s sarcoma were studied through KEGG and signature gene set enrichment analysis.Natural product small molecules targeting these genes were screened using a natural product small molecule database,and Chinese herbal medicines related to the screened natural product small molecules were further investigated.The taste,function,and main indications of the Chinese herbal medicines were analyzed by constructing a natural product small molecule-Chinese herbal medicine network using Cytoscape.The natural product small molecules were ranked using Cytoscape based on drug-like properties(DL)and oral bioavailability(OB)values,and the top four natural product small molecules were selected for cell toxicity experiments to investigate their inhibitory effects on Ewing’s sarcoma cells.Ultimately,natural product small molecule inhibitors that may have an effect on Ewing’s sarcoma treatment were identified.Results:Normalized microarray data sets were obtained,and 1601 genes were found to be significantly up-regulated and 782 genes were significantly down-regulated in Ewing’s sarcoma through differential analysis.GO,KEGG,and GSEA gene enrichment analysis showed that these differentially expressed genes were enriched in pathways related to skeletal and muscle functions,as well as key cellular functions related to cell cycle,myosin binding,cell apoptosis,and TCA cycle.Prognostic analysis revealed that the E2F target pathway had a significant impact on the prognosis of Ewing’s sarcoma.Immune infiltration analysis showed that many T-cell subsets such as CD4 T,CD8 T,γ-δ T were highly infiltrated in tumors,while monocytes and B cells were less infiltrated in tumors.By integrating differential analysis results and survival analysis results,138 significantly up-regulated genes in Ewing’s sarcoma were found to be significantly associated with decreased survival rate,as well as 38 significantly down-regulated genes associated with increased survival rate.Among them,four genes-ASPM,CENPF,CKS2,and DTL-which were up-regulated and significantly associated with decreased survival rate in Ewing’s sarcoma,were selected for validation.Cell proliferation and colony formation experiments showed that silencing these four genes could significantly inhibit the proliferation and colony formation ability of Ewing’s sarcoma cells.Previous studies have shown that high expression of CENPF gene is associated with poor prognosis in various cancers,but its role in Ewing’s sarcoma has not been reported.Pan-cancer analysis showed that CENPF gene is generally highly expressed in various cancers and is associated with poor prognosis.In addition,in terms of immune infiltration,CENPF gene expression is significantly correlated with CD8 T cells and tumor-associated fibroblast infiltration in various cancers.Studies on immune checkpoint genes have shown that CENPF gene expression is significantly correlated with the expression levels of immune checkpoint genes in various cancers.Using RNAi interference technology to construct CENPF knockdown cell lines,the scratch test showed that reducing CENPF expression in Ewing sarcoma cells can significantly inhibit cell migration ability;cell cycle experiments showed that reducing CENPF expression in Ewing sarcoma cells can arrest the cell cycle in the G0/G1 phase,thus affecting the progression of the cell cycle.At the same time,immunoblotting experiments confirmed that the expression of cell cycle-related proteins MCM2,Cyclin D1,Cyclin E1,and C-myc proteins decreased significantly,while the expression of p27 protein increased significantly.Using differential genes from GEO microarray dataset and ICGC RNA-seq transcriptomics dataset,we identified 59 key genes in Ewing’s sarcoma.Gene enrichment analysis showed that these key genes mainly involved cell cycle and E2F target pathways.Then,we searched the Traditional Chinese Medicine Systems Pharmacology(TCMSP)database and constructed a gene-natural product small molecule network using Cytoscape.We selected the top 8 natural product small molecules and constructed a natural product small molecule-Chinese medicine network using the TCMSP database and Cytoscape.These Chinese medicines generally have flavors of pungent,bitter,cold,sweet and neutral,and their main functions include clearing heat and detoxifying,promoting blood circulation and removing blood stasis,and tonifying deficiency and invigorating qi.Finally,through screening based on DL and OB values,we identified that diethylstilbestrol,resveratrol,brazilin,and quercetin may have inhibitory effects on Ewing’s sarcoma cells.We conducted cell toxicity experiments and found that the IC50 values of diethylstilbestrol,resveratrol,brazilin,and quercetin were 56.85 μM,12.33 μM,14.27 μM,and 15.25 μM,respectively.Conclusion:In summary,this study identified the molecular characteristics,immune infiltration,and pathway enrichment features of the transcriptome in Ewing’s sarcoma.ASPM,CENPF,CKS2,and DTL were identified as candidate target genes.Silencing of the CENPF gene inhibited the proliferation,migration,and cell cycle of Ewing’s sarcoma cells,suggesting that CENPF may serve as a target for targeted therapy in Ewing’s sarcoma.Finally,it was confirmed that natural small molecules such as diethylstilbestrol,resveratrol,dyer’s broom extract,and quercetin have inhibitory effects on Ewing’s sarcoma cells.These findings provide insight and a research foundation for understanding the molecular characteristics of Ewing’s sarcoma and identifying molecular targets and natural small molecule inhibitors from traditional Chinese medicine. |
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