| Objective:Triple-negative breast cancer(TNBC)accounts for 15%-20%of the total incidence of breast cancer,and is prone to recurrence and metastasis within 1-3 years.Due to the lack of effective therapeutic targets,TNBC treatment is currently a hot and difficult issue.Tumor microenvironment reprogramming is an important factor affecting tumor invasion and metastasis.A lot of literature has reported that fibroblasts in the tumor microenvironment play an important role in regulating tumor occurrence and development.This study intends to explore the mechanism of exosomes in activating fibroblasts and promoting tumor metastasis by transferring ITGB2 in TNBC using in vitro and in vivo experiments,so as to provide new therapeutic targets for TNBC.Methods:Part Ⅰ: The serum exosomes from 3 patients with TNBC and 3healthy controls were analyzed by proteomics,and the markers ITGB2 and c-Myc relevant to tumor microenvironment regulation were screened.The expression levels of ITGB2 and c-Myc in TNBC exosomes,tissues and cells were verified using Western blotting(WB).Immunohistochemical staining was used to analyze the correlations of ITGB2 and c-Myc in tissues with the prognosis and clinicopathological characteristics of TNBC patients.Part Ⅱ: By constructing TNBC cell lines with a stable and low c-Myc expression,the effects of changed c-Myc expression on the proliferation and invasion of TNBC were analyzed using CCK-8 assay,and cell invasion and metastasis experiments.c-Myc knockout TNBC cells were analyzed by transcriptome sequencing.The mRNAs related to c-Myc expression in TNBC were explored with bioinformatics analyses such as GO analysis,KEGG analysis and protein-protein interaction(PPI)network analysis,and the possible mechanism of c-Myc as a transcription factor regulating gene expression in the tumor microenvironment was analyzed.Part Ⅲ: TNBC cell lines with stable high and low expressions of ITGB2 were constructed.Through co-culture of TNBC exosomes highly and lowly expressing ITGB2 with normal fibroblasts(NF),the mechanism relevant to high ITGB2 expression promoting fibrosis in ITGB2 was analyzed.NF phagocytosis of TNBC exosomes and activation of cancer-associated fibroblasts(CAF)were observed by laser scanning confocal microscopy.The effects of exosomes with ITGB2 expression on the proliferation,migration and invasion of CAF were analyzed using CCK-8 and Transwell assays.By constructing nude mice models of TNBC in the fat pad with high and low ITGB2 expressions,the mechanism of ITGB2 in regulating tumor growth and CAF activation in the tumor microenvironment was analyzed.Results:Part Ⅰ: The proteomic analysis of serum exosomes identified 90 differentially expressed proteins in TNBC patients compared with healthy controls,of which 48 were up-regulated and 42 were down-regulated.They were involved in cell adhesion,positive regulation of biological processes,immune response and other important biological processes.According to the results of biogenic analysis,c-Myc and ITGB2 may be involved in the microenvironment regulation of triple-negative breast cancer tumors.Further RT-PCR and WB verified that the expressions of ITGB2 and c-Myc in TNBC cells,tissues and exosomes increased significantly.Additionally,the high expressions of ITGB2 and c-Myc were positively correlated with the poor prognosis of TNBC.Part Ⅱ: Knockout of c-Myc could significantly inhibit the proliferation and invasion of TNBC cells.This study further analyzed the transcriptome sequencing of the TNBC cell lines after c-Myc knockout.Compared with the control group,276 differentially expressed mRNAs were screened in the c-Myc knockout group by transcriptome sequencing,of which 152 were significantly up-regulated and 124 were significantly down-regulated.Part Ⅲ: In this study,the mechanism of TNBC exosomes activating tumor-associated fibroblasts through ITGB2 was further explored.MDA-MB-231 cell lines with steadily high and low ITGB2 expression were constructed,with MDAMB-231 cell exosomes with high ITGB2 expression enhancing NF activation and its transformation into CAFs after extracting target exosomes and NF co-culturing,thus promoting the proliferation,invasion,and metastasis of CAFs cells.Moreover,ITGB2-knockdown MDA-MB-231 cells demonstrated diminished NF activation.A decrease in the number of endocytosed exosomes in ITGB2-knockdown fibroblasts was observed by confocal laser scanning microscopy,indicating that TNBC exosomes bind to fibroblasts through ITGB2 and initiate membrane fusion before being phagocytosed by fibroblasts.Afterward,the tumor formation experiments in nude mice found that the highest tumor volume emerged after high expression of ITGB2,and tumor growth was significantly inhibited after ITGB2 knockdown,with a lower tumor volume.Furthermore,the down-regulated expression levels of tumor-associated fibroblast markers,i.e.,α-SMA,FAP,and FSP,in the tumor were observed after ITGB2 knockdown,suggesting that ITGB2 knockdown could inhibit CAF activation in the tumor microenvironment.Conclusion:Part Ⅰ: There are significant differences in the protein composition of exosomes between TNBC patients and healthy controls,among which c-Myc and ITGB2 present the most significant differences.It is verified that c-Myc and ITGB2 are significantly highly expressed in TNBC exosomes,cells and tissues,and positively correlated with the poor prognosis of TNBC,suggesting that c-Myc and ITGB2 may be important proteins that regulate and affect the proliferation and invasion of TNBC.Part Ⅱ: Knockout of c-Myc can significantly reduce the proliferation,invasion and metastasis of TNBC.The gene expression profile of c-Myc post-transcriptional regulation was obtained,and it was confirmed that c-Myc may regulate the transcription of ITGB2 gene.Part Ⅲ: Knockout of ITGB2 can significantly inhibit the proliferation,invasion and metastasis of TNBC.The exosomes secreted by TNBC cells can be engulfed and released by NF in the tumor microenvironment through ITGB2,thus activating NF to transform into CAF,and promoting tumor development and metastasis. |
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