The Role And Mechanism Research Of PGRN^-/- TAMs-derived Exosomes In Inhibiting Invasion And Migration Of Breast Cancer Cells

ObjectiveTo investigate the effects of exosomes derived from progranulin（PGRN）deficiency tumor-associated macrophages（TAMs）on invasion and migration of breast cancer cells and their related mechanisms.Methods1.C57BL/6-derived PY8119 breast cancer cells which are PGRN-positive were used to construct mouse orthotopic breast cancer xenograft model on WT and PGRN-deficient(PGRN^-/-)C57BL/6 mice.The tumor volume,lung metastasis and survival rate of WT and PGRN^-/-mice were measured.Flow cytometry was used to compare CD11b⁺F4/80⁺（markers of peripheral blood macrophage）and F4/80⁺CD206⁺（markers of M2 macrophage）cells of WT and PGRN^-/-mouse tumor tissue.2.WT and PGRN^-/-mouse peritoneal macrophages were induced into TAMs in vitro,and the culture supernatant was used to treat C57BL/6-derived breast cancer cells PY8119.Transwell assay,wound healing assay and western blot were used to explore the WT and PGRN^-/-TAMs on breast cancer cell invasion,migration ability and epithelial-mesenchymal transition（EMT）markers epithelial cadherin（E-cadherin）and neuronal cadherin（N-cadherin）expression.3.Exosomes were extracted from WT and PGRN^-/-TAMs by ultracentrifugation,with which treat breast cancer cell PY8119.Transwell assay,wound healing assay and western blot were used to explore the exosomes derived from WT and PGRN^-/-TAMs on breast cancer cell invasion,migration ability and EMT markers E-cadherin and N-cadherin expression.After human monocyte leukemia cells（THP-1 cells）were induced into TAMs,the specific small interfering RNA（siRNA）against PGRN gene was transfected into TAMs.Exosomes were extracted from NC and siPGRN-TAMs,with which treat human breast cancer cells MCF-7and T47D.Transwell assay,wound healing assay and western blot were used to explore the exosomes derived from NC and siPGRN-TAMs on breast cancer cell invasion,migration ability and EMT markers E-cadherin and N-cadherin expression.4.MicroRNA（miRNA）assay was used to analyze the exosomes of NC and siPGRN-TAMs and we found the differentially expressed miRNA of the two groups was miR-5100 and it was verified by real-time fluorescence quantitative PCR.Human breast cancer cells MCF-7 and T47D were treated with exosomes of NC and siPGRN-TAMs,and miR-5100 in breast cancer cells was detected by real-time quantitative PCR.The miR-5100 mimic and inhibitor were transfected into MCF-7 and T47D.Transwell assay,wound healing assay and western blot were used to explore the miR-5100 mimic and inhibitor on breast cancer cell invasion,migration ability and EMT markers E-cadherin and N-cadherin expression.5.The genes regulated by miR-5100 were predicted,selected and verified by real-time fluorescence quantitative PCR and the effect of miR-5100 on the transcriptional activity of the gene was examined by luciferase report assay.Results1.The breast cancer orthotopic tumor formation animal experiments showed that the tumor volumes and lung metastases of PGRN^-/-mice were smaller than those of WT group,and the survival rate was improved.The flow cytometry analysis of mouse tumors showed that the numbers of CD11b⁺F4/80⁺and F4/80⁺CD206⁺cells were decreased in PGRN^-/-mouse tumors.2.WT and PGRN^-/-TAMs culture supernatant was used to treat mouse breast cancer cells PY8119.The results of the transwell assay,wound healing assay and Western blot showed that compared with the WT group,in the PGRN^-/-TAMs group,the invasion and migration ability of breast cancer cells was inhibited,E-cadherin expression was increased and N-cadherin expression was decreased.3.The results of transwell assay,wound healing assay,and Western blot showed that compared with the control groups,exosomes derived from PGRN^-/-TAMs and siPGRN-TAMs caused invasion and migration of breast cancer cells down-regulated,E-cadherin expression up-regulated,and N-cadherin expression down-regulated.4.Real-time quantitative PCR results showed that miR-5100 was raised in exosomes of siPGRN-TAMs compared to the NC group.Human breast cancer cells MCF-7 and T47D were treated with exosomes of NC and siPGRN-TAMs.Real-time quantitative PCR results showed that the breast cancer cells treated with the exosomes derived from siPGRN-TAMs had more miR-5100.The miR-5100 mimic and inhibitor were transfected into MCF-7 and T47D.The results of transwell assay,wound healing assay,and Western blot showed that miR-5100 inhibited the invasion and migration ability of breast cancer cells,increased the expression of E-cadherin and decreased the expression of N-cadherin.5.The results of the luciferase report assay revealed that miR-5100bound to the 3’UTR region of C-X-C motif chemokine ligand 12（CXCL12）and suppressed CXCL12 activity at the level of transcription.Conclusions1.The growth and metastasis of breast cancer of PGRN^-/-mice is inhibited,which is related to TAMs.PGRN^-/-TAMs inhibit the invasion,migration and EMT of breast cancer cells through the exosomes they secret.2.Further exploration of the mechanism shows that miR-5100 of PGRN^-/-TAMs-derived exosomes is up-regulated.MiR-5100 can bind to the 3’UTR region of CXCL12 to regulate its expression,thereby inhibiting the CXCL12/CXCR4 axis,and ultimately inhibiting the invasion,migration and EMT of breast cancer cells.