Study On The Mechanism Of Triple Negative Breast Cancer Cell-derived Exosome ITGB2 To Promote Bone Metastasis In Breast Cancer

Background and objectiveIn 2020,breast cancer became the largest cancer in the world.Breast cancer is the most prone to bone metastasis in female tumors.Once bone metastasis occurs,the median survival rate is only about 3 years.Triple negative breast cancer accounts for15-20% of all breast cancer,and has the characteristics of early age of onset,rapid progress,poor prognosis,easy recurrence,easy metastasis and so on.Therefore,it is of great clinical significance to carry out prevention,diagnosis and treatment for breast cancer patients with the tendency of bone metastasis.Exosomes carry a large number of specific bioinformatics molecules,which are transmitted between cells and microenvironment,and participate in many steps such as the occurrence and development of bone metastases,which is expected to become a new way for the diagnosis and treatment of bone metastases.Therefore,in this study,bioinformatics techniques were used to analyze the specific genes of bone metastasis in triple negative breast cancer,to isolate and extract the exosome(MB231-Exo)derived from MDA-MB-231 in triple negative breast cancer cells,and to explore the mechanism of MB231-Exo and its bone metastasis specific genes in bone metastasis of triple negative breast cancer.The blood samples of patients with breast cancer were collected to analyze the difference of the expression level of bone metastasis related genes between the peripheral blood and the control group,so as to provide theoretical basis for the prevention,early diagnosis and accurate treatment of bone metastasis of breast cancer.Methods1.Exosomes from breast cells and breast cancer cells were extracted by ultra-high speed centrifugal method..The morphology,particle size and structural proteins of Western Blot exosomes were identified by transmission electron microscope,particle size analysis and Western Blot exosome.BCA method was used to determine the protein concentration of exosomes.Nanoparticle tracking analysis and sterility test were used to determine the tracer and sterility status of exosomes in breast cells and breast cancer cells..2.Firstly,the effect of MB231-Exo on the migration of breast cancer cells was confirmed by scratch test combined with exosome inhibitor GW4869,Then,the specific genes up-regulated in bone metastasis of breast cancer were analyzed by GEO database,and the main signal pathways were enriched and obtained.Signal pathway inhibitors were used to verify the effect of signal pathway inhibitors on breast cancer cell migration.At the same time,flow cytometry was used to detect the effects of different treatment groups such as L-NAME and GW4869 on the level of nitric oxide(NO).The expression level of endothelial nitric oxide synthase((endothelial nitric oxide synthase,eNOS/NOS3)and integrin subunit β 2(integrin subunit beta 2)in MB231-Exo was detected by digital PCR,and the role of NOS3 and ITGB2 in MDA-MB-231 migration was further clarified by combining exocrine inhibitor GW4869,NOS signal pathway inhibitor L-NAME and ITGB2 si RNA interference.At the same time,the upstream and downstream genes and main mechanisms of ITGB2 were discussed by means of qPCR and co-IP.Finally,the nude mouse model of bone metastasis of MDA-MB-231-luc breast cancer was established and intervened with exocrine MB231-Exo,exosome inhibitor GW4869 and NOS signal pathway inhibitor L-NAME.The real-time metastasis of MDA-MB-231 cells in the mouse model was observed by in vivo imaging,and the cell migration and proliferation were observed by immunohistochemistry.The expression levels of NOS3,ITGB2 and upstream and downstream genes in tumor and bone tissue were verified by qPCR method.3.Firstly,the peripheral blood samples of 5 healthy people,5 patients with primary triple negative breast cancer without metastasis and 5 patients with bone metastasis of breast cancer were collected from the Breast Department of Affiliated Cancer Hospital of Zhengzhou University in 2020.The plasma exosomes were extracted and the expression levels of breast cancer bone metastasis specific genes NOS3,ITGB2 and S100 P in the plasma exosomes of the above researchers were detected by digital PCR.Using TCGA database,bioinformatics analysis was carried out based on the clinical data related to breast cancer,focusing on the expression of S100 P,NOS3 and ITGB2 in breast cancer and other tumor tissues,and analyzing the relationship between S100 P,NOS3 and prognosis of breast cancer.The clinical data of 64 patients with triple negative breast cancer in the Breast Department of Affiliated Cancer Hospital of Zhengzhou University from January to December 2016 were analyzed retrospectively.the plasma exosomes from peripheral blood were extracted and the relative expression levels of S100 P and ITGB2 in the exosomes were detected.Combined with clinical data,ROC curve was established to analyze the sensitivity and specificity of predicting bone metastasis.Results1.The exosomes were successfully extracted and purified from the supernatant of breast cancer cells by ultrahigh speed centrifugation.Transmission electron microscopy showed that the diameters of exosomes derived from breast cancer cells MCF-7,MDA-MB-231 and normal breast epithelial cells MCF10 A were between 30 and 150 nm,showing membranous vesicles with tea-like structure,and the average particle sizes of exosomes of MCF-7,MDA-MB-231 and MCF10 A were 79.94 nm,73.65 nm and 79.81 nm,respectively.Western Blot assay confirmed that all exosomes expressed exosome structural protein TSG101.The results of exosome tracer assay showed that the binding ability of MB231-Exo to cells was stronger than that of MCF-7-Exo and MCF10A-Exo.2.It was found that MB231-Exo derived from MDA-MB-231 could promote the migration of breast cancer cells in vitro,and exosome inhibitor GW4869 could inhibit the migration-promoting effect of MB231-Exo.At the same time,296 significant differential genes specific to bone metastasis of breast cancer were obtained by analyzing GEO database,and significant changes in eNOS activation and amebodial-type cell migration signal pathways were found by enrichment analysis.The results of NO flow assay showed that NOS inhibitor L-NAME could reduce the exosome production and reverse the migration of MDA-MB-231,indicating that eNOS activation can regulate the tumor metastasis promoting effect of MB231-Exo.NOS3 and ITGB2 genes closely related to bone metastasis were screened by analyzing the genes of eNOS activation and amebodial-type cell migration pathway.The expression of NOS3 and ITGB2 in MDA-MB-231 cells and exosomes was higher than that of MCF-10 A and MCF-7,L-NAME in the control group by digital PCR.While down-regulating the expression of NOS3,it also down-regulated the expression of ITGB2,indicating that ITGB2 may be regulated by NOS3.Further analysis showed that the expression of ITGB2 transcription factor RUNX2 could also be inhibited by L-NAME.Scratch test showed that siITGB2 could inhibit the migration of MDA-MB-231 cells,indicating that ITGB2 may be the key molecule for NOS to promote migration.QPCR results showed that NOS3 was positively correlated with the expression of ITGB2 and its transcriptional RUNX2 and downstream molecule FAK.After L-NAME inhibited NOS3,the expression levels of RUNX2,ITGB2 and FAK decreased.Co-IP results show that ITGB2 can directly bind to FAK,which is the key molecule in regulating tumor metastasis.These results suggest that NOS can regulate the secretion of MB231-Exo,inhibition of NOS can down-regulate the expression of RUNX2 and target gene ITGB2,and ITGB2 can directly bind to FAK protein to promote MDA-MB-231 migration.In animal experiments,in vivo imaging and immunohistochemical results showed that both L-NAME and GW4869 could inhibit the proliferation and metastasis of MDA-MB-231 cells,while the control group had the fastest metastasis and liver metastasis.the results of qPCR confirmed that L-NAME down-regulated the level of NOS3,and the expression of RUNX2 and its target gene ITGB2 and tumor-related gene FAK were also down-regulated in tumor and bone tissue.The results also showed that the expression level of S100 P in MDA-MB-231 was higher than that in MCF-7 and MCF-10 A groups,and the same phenomenon was observed in the corresponding exosomes.L-NAME had no regulation on the secretion of S100 P.3.Digital PCR detection showed that S100 P and ITGB2 were highly expressed in plasma exosomes of patients with bone metastasis of breast cancer,and there was significant difference compared with the control group,especially S100P(P < 0.001).There was no significant difference in NOS3 among the three groups.TCGA database analysis showed that the expression of S100 P in breast cancer tissues was higher than that in paracancerous tissues,and was related to prognosis.The clinical data showed that there was significant difference between the relative expression of S100 P and histological grade,pathological stage,lymph node metastasis and bone metastasis,but not with age,while there was significant difference between the relative expression of ITGB2 and pathological stage,lymph node metastasis and bone metastasis(P < 0.05),but not with age and histological grade.According to the occurrence of bone metastasis within 3 years,the two groups were divided into two groups to establish ROC curve.The ROC curve showed that the diagnostic value of ITGB2 in breast cancer bone metastasis(AUC=0.848,sensitivity: 81%,specificity:86%)was higher than that of S100P(AUC=0.832,sensitivity: 71.4%,specificity:95.3%).The AUC value of ITGB2 and NOS3 combined diagnosis was the highest(AUC=0.900,sensitivity: 90.5%,specificity 86%).The results showed that the expression of ITGB2 and S100 P in plasma exosomes had high sensitivity and specificity in the diagnosis of bone metastasis of triple negative breast cancer within 3years.Conclusion:1.Triple-negative breast cancer cell-derived exosome MB231-Exo obtained by hypervelocity centrifugation is easy to be taken up by recipient cells and is suitable for subsequent studies.2.The exosomes of triple-negative breast cancer with high metastatic potential promotes the growth of breast cancer transplant tumor through eNOS activation in vivo and in vitro.NOS inhibitor L-NAME can inhibit the production of MB231-Exo in exosomes of triple-negative breast cancer cells,thus effectively inhibit its role in promoting the growth and metastasis of breast cancer.The main acting molecule in the exosomes of triple negative breast cancer cells is ITGB2.The signal pathway mechanism may be that NOS upregulates the expression of target gene ITGB2 by up-regulating the transcription of RUNX2,and then ITGB2 binds to the signal protein FAK to promote the invasion and bone metastasis of breast cancer cells.3.The expression of S100 P in breast cancer is higher than that in paracancerous tissues and is related to prognosis,suggesting that S100 P may be a potential tumor marker.The expression of S100 P and ITGB2 was up-regulated in plasma exosomes of patients with bone metastasis of breast cancer.The expression levels of ITGB2 and S100 p in plasma exosomes have high sensitivity and specificity for the diagnosis of bone metastasis in patients with triple-negative breast cancer within 3 years,and can be used for the auxiliary diagnosis of bone metastasis in triple-negative breast cancer.