Exosomes Of Mouse Melanoma Cells Promote Fibroblast Migration By Regulating Rac1 Protein

Cell-to-cell interaction depends on intercellular communication and signal regulation.In addition to direct secretion of chemical signals,intercellular contact and intercellular gap junction,recent studies have found that exosomes released by cells are also an important medium for intercellular communication.Exosomes transfers proteins,nucleic acids,lipids and other information molecules of the donor cells to the recipient cells,thus regulating the biological function of the receptor cells.After the normal stromal cells in the tumor microenvironment take up metastatic tumor cell-derived exosomes,they can reprogram and transform into tumor precursor cells under the regulation of donor cells information,showing the potential of invasion and migration,which lays a foundation for tumor cell migration and colonization.Fibroblasts are a kind of important stromal cells in tumor microenvironment,which Fibroblasts can assist in the migration and colonization of tumor cells through transformation into tumor precursor cells.The previous study of our group revealed that mouse melanoma B16-F10 cell exosomes can induce mouse embryonic fibroblasts(MEF)to express invasive characteristics,indicating that melanoma cell exosomes may promote the transformation of fibroblasts into tumor precursor cells.Rac1 protein is an important molecule in the regulation of intracellular information,which plays an important role in the invasion and migration of tumor cells,mainly by reorganizing the cytoskeleton depending on its expression level and its different distribution in the cells,promoting the extension of lamellipodia and membrane ruffling,and promoting cell invasion and migration.In this paper,mouse melanoma B16-F10 cells and mouse embryonic fibroblast MEF cells were used as materials to study the effect of B16-F10 cell exosomes on the migration ability of MEF cells and its mechanism,to further verify the possibility of melanoma cell exosomes promoting the transformation of fibroblasts into tumor precursor cells,and to provide a new idea for explaining the mechanism of tumor cell invasion and migration.Objectives1.To establish a co-culture system of B16-F10 exosomes and MEF cells,to analyze the effect of B16-F10 exosomes on the migration ability of MEF cells,and to provide evidence that metastatic tumor exosomes affects the transformation of fibroblasts to tumor precursor cells.2.On the basis of the established co-culture system of B16-F10 exosomes promoting MEF cells migration,the effects of B16-F10 exosome on the expression of Racl protein and its different distribution in MEF cells was analyzed,and the partial mechanism of metastatic tumor exosome promoting fibroblast invasion and migration were explained.Methods1.Employing highly metastatic cell line B16-F10 and primary MEF cells as the research objects,the exosomes of B16-F10 exosomes were obtained by ultracentrifugation.The typical shape of the exosomes was observed by negative staining under transmission electron microscope,the size distribution of the exosomes detected by Nonoparticle tracking analysis(NTA),and the tumor susceptibility gene 101(Tsg101)and tyrosine-related protein 2(Tyrp2)were identified by Western-blot,identify B16-F10 exosomes obtained by ultracentrifugation.2.The co-culture system of B16-F10 exosomes and MEF cells was established,and set the groups of the 0 h,12 h,24 h and 36 h,the ability of MEF cells took in PKH-26-labeled B16-F10 exosomes in vitro was detected by laser confocal microscope and flow cytometry,respectively.Employing the MEF cell group as the control group and co-culture group as experimental group,set the groups of the 0 h,12 h,24 h and 36 h,the effect of B16-F10 exosomes on the migration of MEFcells was determined by transwell assay.Western-blot assay was used to detect the level of E-caderhin protein expression of B16-F10 exosomes on MEF cells in co-culture system.3.On the basis of the established co-culture system which B16-F10 cells exosomes promote MEF cell migration,the transcriptional level of RaclmRNA in MEF cells affected by B16-F10 exosomes was detected by RT-PCR assay.The expression level of Rac1 protein in MEF cells affected by B16-F10 exosomes was detected by western-blot assay.The expression level of Racl and its different distribution in MEF cells affected by B16-F10 exosomes was detected by cellular immunofluorescence staining.Results1.The exosomes of B16-F10 cells obtained by ultracentrifugation showed typical saucer-like morphology,the particle size was distributed among 164 nt～255 nm,and expressed melanoma characteristic protein Tyrp2 and exosomes characteristic protein Tsg101.2.B16-F10 exosomes co-culture with MEF cells for 0 h,non-PKH26-labled exosomes gathered in MEF cells,after co-cultured to 12 h,a small amount of B16-F10 exosomes gathered at the cytoplasmic edge of MEF cells,and a large number of exosomes gathered in the cytoplasm and around the nucleus of MEF cells at 24 h and 36 h after co-culture.And the uptake rate of PKH26-labeled exosomes by MEF cells co-culture for 12 h,24 h and 36 h was increased significantly higher than that of co-culture for 0 h(P<0.01).The results of migration assa showed that the migration ability of MEF cells co-cultured for 12 h,24 h and 36 h was significantly higher than that of co-culture for 0 h(P<0.01).At the same time,the expression of E-caderhin protein in MEF cells descreased significantly(P<0.05,P<0.01).3.In the co-culture system,compared with co-culture for 0 h,the transcript level of RaclmRNA in MEF cells increased significantly after co-culture for 24 h,and the expression of Racl protein increased gradually in MEF cells after co-culture for 24 h and 36 h(P<0.05,P<0.01).At the same time,Racl protein expression showed a sequential position change,distributed in different degrees in the cell membrane,cytoplasm and nucleus of MEF cells.conclusion1.Through the establishment of a co-culture system of mouse melanoma B16-F10 exosomes and MEF cells,it was determined that the migration ability of MEF cells was increased after taking B16-F10 exosomes,indicating that the that metastatic tumor cells affect the transformation of fibroblasts to tumor precursor cells by transporting exosomes.2.On the basis of the co-culture system of B16-F10 exosomes promoting MEF cells migration in vitro,it was determined that MEF cell uptake of B16-F10 cell exosomes promote the expression level of Racl protein,moreover,the expression position of Racl protein changes in a time-dependent manner,and it’s distribution in different degrees in the plasma membrane,cytoplasm and nucleus,indicating that B16-F10 cell exosomes promotes MEF cells migration by regulating the expression level of Racl protein and its different distribution in cells.