| Objective(s):Epithelial ovarian cancer has a poor prognosis due to the difficulty in early diagnosis,high recurrence and metastasis.Exosomes and their contents are closely related to the extravasation,immune escape and metastasis of malignant tumors,which can be used as potential markers for early diagnosis of malignant tumors.Tumor microenvironment is the cellular environment for tumor survival,which can promote tumor progression and heterogeneity.Tumor-associated macrophages are important components of tumor microenvironment,which can promote angiogenesis,invasion and migration of malignant tumors.The aim of this study is to analyze the differential expression profiles of exosomes from different ovarian tissues,screen out the differential exosomal mRNAs,explore the regulatory effect of ovarian cancer tissue-derived exosomes on macrophage polarization,and provide experimental data for the early diagnosis,occurrence and development of epithelial ovarian cancer.Methods:1.The specimens of patients who underwent oophorectomy in the Department of Gynecology,Yunnan Cancer Hospital from January 2021 to January 2022 were collected.The ovarian specimens from patients who required ovarian resection,clinical diagnosis and postoperative pathological diagnosis of uterine leiomyoma,adenomyosis,etc.were included in the normal ovarian tissue collection.2.Primary epithelial ovarian cancer cells were isolated.Ovarian tissue-derived exosomes were isolated and extracted by ultracentrifugation(normal ovarian tissue group,benign ovarian tumor group,borderline ovarian tumor group,early ovarian malignant tumor,advanced ovarian malignant tumor),identified by NTA and flow cytometry,and the surface marker CD9 of exosomes was detected by flow cytometry.3.High-throughput sequencing of ovarian cancer exosome transcriptome was used to compare and analyze the differential expression of exosomal mRNA in each group.GO,KEGG pathway and other bioinformatics analysis were used to screen out the differential genes.Western Blot(WB)was used to detect the expression level of THBS1 gene in exosomes of different groups.4.THBS1 was knocked down,and the expression levels of M1 macrophage marker CD86 and M2 macrophage marker CD206 were detected by WB.5.CCK8 assay and cell scratch assay were used to investigate the effect of ovarian cancer exosomes on regulating macrophage polarization and promoting tumor proliferation and migration through THBS1 gene.Results:1.Under transmission electron microscope,there was no significant difference in the shape and size of ovarian exosomes from different sources,and they were disc-shaped cystic structures.The average particle size of ovarian tissue exosomes from different sources was 176.3 nm,165.9 nm,174.2 nm,151.7 nm,155.8 nm in normal ovarian tissue,benign ovarian tumor,borderline ovarian tumor,early malignant ovarian tumor and late malignant ovarian tumor,respectively.The average concentration was 1.5E+11 Particles/ml,7.2E+10 Particles/ml,1.6E+11 Particles/ml,1.6E+11 Particles/ml,6.0E+9 Particles/ml,respectively.There was no significant difference in particle size and concentration among the groups(P < 0.05).Flow cytometry showed that the exosomal marker CD9 was positive in all groups,and there was no significant difference in the positive rate of CD9 among groups(P < 0.05).2.The results of high-throughput sequencing showed that the gene composition of early stage and advanced stage of ovarian malignant tumors was similar,and the gene composition of benign and borderline ovarian tumors was similar.There were 26 differentially expressed mRNAs between the ovarian benign tumor group and the normal tissue group,including 21 up-regulated mrnas and 5 down-regulated mrnas.There were 20 differentially expressed mRNAs between the ovarian borderline tumor group and the normal tissue group,of which 18 were up-regulated and 2 were down-regulated.There were 349 differentially expressed mRNAs between the early ovarian cancer group and the normal tissue group,of which 199 mrnas were up-regulated and 150 mrnas were down-regulated.There were 741 differentially expressed mRNAs between the advanced ovarian cancer group and the normal tissue group,including 501 up-regulated mrnas and 240 down-regulated mrnas.3.GO analysis showed that the exosomes-related differential mRNAs were mainly involved in multicellular biological processes,cell proliferation and differentiation,cell migration,anatomical structure development,cell response to chemical stimulation,negative regulation of cell processes,cell adhesion,SMAD protein complex,glycosaminoglycan binding molecules,extracellular matrix organization,angiogenesis,etc.4.KEGG analysis showed that the differentially expressed exosome-related mRNAs in each group were mainly involved in PI3K-Akt signaling pathway,p53 signaling pathway,TNF signaling pathway,TGF-β signaling pathway,human papillomavirus infection,etc.5.By drawing Venn diagram,we obtained 16 differentially expressed genes in benign group,early malignant group and late malignant group.Through PPI analysis,we predicted differentially expressed gene mRNAs and interaction network,and finally identified the differentially expressed THBS1 gene.6.The results of WB showed that THBS1 was expressed in normal tissue group,benign ovarian tumor group,borderline ovarian tumor group,early malignant tumor group and late malignant tumor group.Among them,the expression levels of early malignant tumor group and late malignant tumor group were significantly higher than those of normal tissue group,benign tumor group and borderline tumor group(P <0.001).7.WB results showed that after knocking down the differential gene THBS1,the protein expression level of CD206,a marker of M2 macrophages,was significantly lower than that of the control group and the blank vector group(P < 0.001),and the protein expression level of CD86,a marker of M1 macrophages,was significantly higher than that of the control group and the blank vector group(P < 0.001).CCK-8assay showed that the proliferation activity of THBS1 knockdown cells was significantly lower than that of the blank vector group and the control group.Cell scratch assay showed that the migration ability of THBS1 knockdown cells was lower than that of the blank vector group and the control group.Conclusion(s):1.There was no significant difference in the concentration and morphology of exosomes from different ovarian tissues.2.The composition of exosomes derived from different ovarian tissues was different,among which the expression of mRNAs in the early stage and advanced stage of ovarian cancer was significantly different from that in the normal ovarian tissue.The differential mRNAs were mainly involved in p53 signaling pathway,PI3K-Akt signaling pathway,TGF-β signaling pathway,etc.3.Ovarian cancer-derived exosomal THBS1 gene promotes the polarization of macrophages to M2 type.After THBS1 gene knockdown,macrophages were polarized to M1 type.4.Ovarian cancer-derived exosomal THBS1 gene can promote the proliferation and migration of ovarian cancer cells.Therefore,the mRNA of ovarian cancer exosomal THBS1 gene may be a potential molecular marker for early diagnosis and promotion of tumor proliferation and migration.At present,the mechanism by which ovarian cancer exosomes induce macrophage polarization to M2 type through THBS1 gene is still unclear and needs to be further studied. |
PDF Full Text Request