| Background:Breast cancer is one of the most common tumors in women.Most patients have poor prognosis due to distant organ metastasis,and the molecular mechanism of breast cancer metastasis still needs to be further explored.The establishment of pre-metastatic niche(PMN)provided a local microenvironment for the colonization and growth of circulating tumor cells and promoted the distant metastasis of tumor cells.Exosomes are extracellular vesicles 30-100nm in diameter that carry nucleic acids and proteins.Exosomes play a key role in the formation of PMN.Exosomes can form PMN that is beneficial to the growth of tumor cells by changing the phenotype and physiological function of distant stromal cells.Our previous study found that compared with breast cancer patients without metastasis,peripheral blood exosomes miR-122-5p,miR-193A-5p,miR-320b and miR-375 in breast cancer patients with metastasis were significantly upregulated.Objective:To investigate the specific molecular mechanisms by which breast cancer exosomes miR-122-5p,miR-193A-5p,miR-320b and miR-375 may promote distant metastasis of breast cancer cells.Methods:Exosomes were collected by ultracentrifugation and identified by transmission electron microscopy.The uptake of fibroblast WI-38 exosomes was then verified using a PKH67 fluorescent dye.Transwell migration assay was used to test the regulation effect of exosomes or miRNAs on the migration ability of MDA-MB-231 cells.Western Blot analysis was used to verify the activation effect of exosomes on WI38 cells,and transmission electron microscopy was used to discover the influence of exosomes on the morphology of Golgi in WI-38.ELISA was used to detect The regulation effect of MDA-MB-231 exosomes on the expression of secreted proteins in WI-38 cells.The expression levels of miR-122-5p,miR-193A-5p,miR-320b and miR375 in the cytoplasm and exosomes of cells were detected by RT-qPCR.The effects of miR-122-5p,miR-193A-5p,miR-320b and miR-375 on the expression of the protein secreted by WI-38 were preliminarily investigated by ELISA using miRNA mimic.Then,specific miRNA mimic/inhibitor was used to verify the effect of MDA-MB-231 exosome miRNA on the expression of secreted protein in WI-38 cells by ELISA.Transwell was used to examine the effect of MDA-MB-231 exosomes miRNA on the secretary levels of chemokines in WI-38 cells to promote the migration of MDA-MB231.The direct targeting of miRNA to downstream targets was verified by dual luciferase reporter genes.Western Blot and ELISA confirmed the regulation effect of MDA-MB-231 exosome miRNA on WI-38 intracellular target and its downstream molecules.Results:Transmission electron microscopy showed that exosomes were disk-like in appearance,with a diameter of about 100nm.Western Blot verified the extracted exosomes expression marker proteins TSG101 and CD81.Fluorescence microscopy observed that WI-38 cells could take up PKH-labeled exosomes from MCF10A and MDA-MB-231 cells.Transwell cell experiments confirmed that MDA-MB-231 exosomes promoted the migration of MDA-MB-231 cells by affecting WI-38 cells.Western Blot analysis showed that MDA-MB-231 exosomes could increase the expression of α-SMA in WI-38 cells,and transmission electron microscopy observed that MDA-MB-231 exosomes changed the morphology of Golgiosomes in WI-38 cells.ELISA results showed that Mda-MB-231 exosomes could increase the secretion of MCP-1,SDF-1 and laminin,and decrease the secretion of fibroconnectin in WI-38 cells.RT-qPCR results showed that the expression levels of miR-122-5p,miR-193A-5p,miR-320b and miR-375 in MDA-MB-231 cells were low,while the levels in the exosomes were high.ELISA results showed that miR-122-5p mimic could increase the secretion of MCP-1,SDF-1 and laminin in WI-38 cells,while decrease the secretion of fibronectin.ELISA results confirmed that miR-122-5p,an exosome of MD-MB-231,could increase the secretion of MCP-1,SDF-1 and laminin in WI-38 cells,while decrease the secretion of fibronectin.Transwell cell migration assay showed that the MDA-MB-231 exosome miR-122-5p promoted the migration of MDA-MB-231 cells by increasing the expression of MCP-1 and SDF-1 in WI-38 cells.Dual-luciferase reporter gene results showed that miR-122-5p could directly target MKP-2.Western Blot and ELISA results showed that the MDA-MB-231 exosome miR-122-5p downregulated MKP-2 in WI-38 cells,and increased the expression levels of MCP-1 and SDF-1 by regulating ERK and JNK signaling pathways.Conclusions:MDA-MB-231 exosome miR-122-5p can target MKP-2 and increase the expression levels of MCP-1 and SDF-1 in WI-38 cells through ERK and JNK pathways.MCP-1 and SDF-1 are able to enhance the migration ability of MDA-MB231 cells,thus promoting the metastasis of breast cancer cells. |
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