The Role And Mechanism Of Long Non-coding RNA FOXP4-AS1 In Ewing Sarcoma And Tumor Microenvironment

Background:Ewing sarcoma is a primary and highly malignant bone tumor originating from the bone marrow.It mainly occurs in children and adolescents,with an incidence rate of 1 in 1.5 million,with the highest incidence in people aged 15 years.Ewing sarcoma can occur in any part of the body,but the most common disease sites are the pelvis and the proximal end of the long bone.The disease has a high degree of malignancy,a short course of the disease,and a poor prognosis,with a 5-year survival rate of 70%to 80%.30%of the patients had significant metastases at the time of diagnosis,and the 5-year survival rate for metastatic tumors was only 30%.The Ewing sarcoma treatment includes simple surgery,radiotherapy,and chemotherapy,but the effect is not ideal.Therefore,understanding the mechanism of the occurrence and development of ES will help to find new ways of early diagnosis and treatment,improve the survival rate and reduce the recurrence rate,and solve the critical scientific problems in the treatment of ES.LncRNA is a non-coding RNA,a transcript of more than 200 nucleotides in length,involved in the critical biological processes of physiological development and disease occurrence.Dysregulation of lncRNA expression plays a vital role in malignant transformation of cells,including maintenance of cell growth and proliferation,promotion of metastasis and invasion,induction of angiogenesis,and inhibition of apoptosis,et al.They are closely related to the occurrence,development,and prognosis of tumors.LncRNAs are also diversified in subcellular distribution and have different biological functions in different subcellular locations.In the nucleus,it can act as a molecular scaffold,assist in alternative splicing,and regulate chromosome structure,while in the cytoplasm,it can regulate translation,promote or inhibit mRNA degradation,adsorb miRNA(act as competitive endogenous RNA,ceRNA).Thus,researching the function and expression of lncRNAs will contribute to understanding the pathogenesis,diagnosis,treatment,and prognosis of ESTumor microenvironment(TME)refers to the surrounding micro-environment of Tumor cells,including the surrounding blood vessels,immune cells,fibroblasts,bone marrow-derived inflammatory cells,extracellular matrix,various cytokines,and chemokines,etc.,which is a complex and comprehensive system.Studies have shown that lncRNAs play an essential role in tumor cells and can be wrapped by extracellular vesicles and delivered to TME to participate in inter-cellular information transmission,affect TME composition,and then regulate the occurrence and development of tumors.Therefore,the study of the functional mechanism of lncRNA in ES and its microenvironment is of great significance for finding new ES diagnosis and treatment methods.In summary,this study intends to study the functional mechanism of lncRNA in the occurrence and development of ES from both the tumor cell and the tumor microenvironment.Objective:This study aimed to find new prognostic lncRNA markers for ES and study the functional role of lncRNA in ES and its influence on the tumor microenvironment.The study is divided into the following five parts:1.Screening and clinical significance of long non-coding RNA FOXP4-AS1;2.Effects of FOXP4-AS1 on proliferation,migration,and invasion of Ewing sarcoma cell lines;3.Bioinformatics analysis of the potential mechanism of FOXP4-AS1 in Ewing's sarcoma;4.FOXP4-AS1 regulates the proliferation,migration,and invasion of Ewing's sarcoma through miR-298/TMPO pathway;5.Functional study of FOXP4-AS1 in Ewing's sarcoma tumor microenvironmentThe above research results help clarify the function and mechanism of prognostic lncRNA in ES and have excellent clinical relevance and value in predicting tumor prognosis,which can provide a new idea for the diagnosis,treatment,and clinical transformation of ES.Part ? Screening and clinical significance of long non-coding RNAFOXP4-AS1 Methods:1.Prognostic-related lncRNAs of Ewing sarcoma were screened by lasso-Cox regression.2.Kaplan-Meier survival analysis was used to screen the prognostic IncRNAs differentially expressed.3.The expression of FOXP4-AS1 in Ewing's sarcoma and 33 kinds of tumors was analyzed by GEO,TCGA,GTEX data,and RT-qPCR.Results:1.FOXP4-AS1 is a poor prognostic factor of Ewing sarcoma;2.The expression of FOXP4-AS1 was significantly increased in Ewing sarcoma tissues and cell lines;3.FOXP4-AS1 expression is elevated in various malignant tumorsPart ? Effects of FOXP4-AS1 on proliferation,migration,and invasion of Ewing's sarcoma cell linesMethods:1.The effect of knockdown or overexpression of FOXP4-AS1 on the proliferation of Ewing sarcoma cells was detected by CCK-8 assay;2.Transwell migration and invasion assay was used to detect the effects of knockdown or overexpression of FOXP4-AS1 on migration and invasion of Ewing's sarcoma cells;3.After knockdown or overexpression of FOXP4-AS1,Ewing's sarcoma cells'proliferation and tumor-forming ability were detected by subcutaneous tumor-forming assay in nude mice.Results:FOXP4-AS1 could promote proliferation,migration,and invasion ability of Ewing sarcoma cell lines,then promote Ewing sarcoma's occurrence and development.Part ? Bioinformatics analysis of the potential mechanism of FOXP4-AS1 in Ewing sarcomaMethods:1.Screening the genes significantly related to FOXP4-AS1,and using GO,Hallmark Gene sets enrichment analysis,and protein interaction network analysis to screen the functions and pathways significantly related to FOXP4-AS1.2.Combined screening of FOXP4-AS 1-related signaling pathways by GSVA and GSEA.3.The subcellular localization and potential molecular function of FOXP4-AS1 were analyzed by AGO2-IP assay.4.The FOXP4-AS1 ceRNA network was constructed,and the core genes related to the enrichment pathway were screened and the downstream target genes of FOXP4-AS1 were determined by combining the enrichment analysis results of the signaling pathway.Results:1.In Ewing sarcoma cells,FOXP4-AS1 is mainly localized in the cytoplasm and has the function of "ceRNA."2.FOXP4-AS1 may regulate the pathogenesis of Ewing sarcoma as a ceRNA by sponging miR-298 to upregulate TMPO.Part ? FOXP4-AS1 regulates the proliferation,migration,and invasion of Ewing's sarcoma through miR-298/TMPO pathwayMethods:1.The binding sites of miR-298 with FOXP4-AS1 and TMPO were verified by Dual Luciferase Reporter Gene Assay2.In the stable cell lines with low expression of FOXP4-AS1,miR-298 mimics,and miR-298-inhibitor were transfected,respectively.The changes in proliferation,migration,and invasion ability of Ewing's sarcoma cells were detected by CCK-8 and Transwell migration and invasion assay.3.MiR-298 mimics and miR-298 mimics+pcDNA-TMPO were transfected into Ewing's sarcoma cells,respectively,and RT-qPCR and Western blot detected the expression of TMPO.Finally,the proliferation,migration,and invasion ability of Ewing's sarcoma cells were detected by CCK-8 and Transwell migration and invasion experiments.Results:FOXP4-AS1 regulates the expression of TMPO by sponging miR-298,thus promoting the proliferation,migration,and invasion of Ewing sarcomaPart ? Functional study of FOXP4-AS1 in Ewing's sarcoma tumor microenvironmentMethods:1.The relationship between FOXP4-AS1 and 22 immune cell infiltration levels in Ewing's sarcoma tumor microenvironment was analyzed by the Cibersort method2.Three kinds of macrophages,M0,M1,and M2,were constructed by THP-1 cells to detect the expression of FOXP4-AS13.FOXP4-AS1 was knockdown and overexpression respectively in M0 macrophages,and then IL-4/IL-13 was used to induce M0 polarization to M2 macrophages.After 24 hours,M1 and M2 cell markers were detected to evaluate cell polarization.4.The expression of FOXP4-AS1 in extracellular vesicles was detected,and the phagocytosis of extracellular vesicles in macrophages was detected by PKH26 vesicle labeling assay5.Extracellular vesicles of overexpressed FOXP4-AS1 cells were extracted for co-culture with M0 macrophages.After 48 hours,M2 cell markers were detected by RT-qPCRResults:FOXP4-AS1 can be delivered to tumor-associated macrophages by Ewing sarcoma-derived extracellular vesicles and can induce M2 polarization of macrophages.Conclusion:1.FOXP4-AS1 is a poor prognostic biomarker for Ewing sarcoma.2.FOXP4-AS1 can promote the proliferation,migration,and invasion of Ewing's sarcoma.3.FOXP4-AS1 positively regulated TMPO by sponging miR-298 and promote Ewing sarcoma tumorigenesis.4.FOXP4-AS1 can be delivered to tumor-associated macrophages by extracellular vesicles and induce M2-type polarization.