Study The Effects Of BM-MSCs-derived Exosomes On EMT-Like Phenotype,Stemness And Invasion Of Myeloma Cells

Objective: 1.To investigate the effects of bone marrow mesenchymal stem cells(BM-MSCs)-derived exosomes of multiple myeloma(MM)patients with or without extramedullary disease(EMD)on the EMT-like phenotype of MM cell lines and primary myeloma cells in vitro.2.To investigate the effects of bone marrow mesenchymal stem cells(BM-MSCs)-derived exosomes of multiple myeloma(MM)patients with or without extramedullary disease(EMD)on the stemness of MM cell lines in vitro.3.To investigate the effects of bone marrow mesenchymal stem cells(BM-MSCs)-derived exosomes of multiple myeloma(MM)patients with or without extramedullary disease(EMD)on the invasion of MM cell lines and primary myeloma cells in vitro.Methods: 1.Isolation,culture of bone marrow mesenchymal stem cells and primary myeloma cells.2.Exosomes were isolated by ultracentrifugation from the cell culture supernatant of BM-MSCs.3.Characterization of BM-MSCs-derived exosomes by transmission electron microscopy;Western blot analyses of exosomal markers CD81 and negative protein Cytochrome C in BM-MSCs lysates and BM-MSCs-derived exosomes.4.Exosomes were labeled with fluorescent dye PKH-67,and then co-cultured with myeloma cells;then stained nucleus of myeloma cells with DAPI,and observed the uptake of BM-MSCs-derived exosomes by myeloma cells using confocal microscopy.5.The m RNAs expression levels of the EMT(Vimentin,Twist)and stemness(NANOG,OCT-4)-related markers in myeloma cells co-cultured with BM-MSCs-derived exosomes of MM patients with or without EMD were detected by Real-Time PCR.6.The proteins expression levels of the EMT-related markers(E-Cadherin,N-Cadherin,Vimentin,?-Catenin)in control myeloma cells(LP-1,U266,primary myeloma cells),myeloma cells co-cultured with BM-MSCs-derived exosomes of MM patients with or without EMD were detected by Western Blot.7.The invasion of control myeloma cells(LP-1,U266,primary myeloma cells),myeloma cells co-cultured with BM-MSCs-derived exosomes of MM patients with or without EMD were detected using Transwell Invasion Assay.8.Study the difference in mi RNAs between BM-MSCs-derived exosomes of MM with and without EMD by mi RNAs sequencing and validate significantly differential mi RNAs between BM-MSCs-derived exosomes of MM with and without EMD by Real-Time PCR.Results: 1.BM-MSCs-derived exosomes showed a diameter of about 100 nm under transmission electron microscopy.These exosomes had obvious membranes,whose three-dimensional structure was clear.The shape of these exosomes was tea tray-like or concave hemisphere.Western blot showed that these exosomes expressed CD81 and did not express Cytochrome C.2.U266,LP-1 cell lines and primary myeloma cells had significant uptake of BM-MSCs-derived exosomes.3.Real-Time PCR showed that BM-MSCs-derived exosomes of MM patients with EMD increased m RNAs expression of EMT(Vimentin,Twist)and stemness(NANOG,OCT-4)-related markers of U266,LP-1 cell lines.4.Western bolt showed that BM-MSCs-derived exosomes of MM patients with EMD increased proteins expression of EMT-related markers(N-Cadherin,Vimentin,?-Catenin)and decreased protein expression of E-Cadherin of U266,LP-1 cell lines and primary myeloma cells.5.Transwell Invasion Assay showed that BM-MSCs-derived exosomes of MM patients with EMD enhanced invasion of U266,LP-1 cell lines and primary myeloma cells.6.Real-Time PCR showed that mi R-199 a and mi R-197 are increased in the BM-MSCs-derived exosomes of MM with EMD compared with that without.Conclusion: LP-1,U266 cell lines and primary myeloma cells were co-cultured with BM-MSCs-derived exosomes,and we observed myeloma cells had significant uptake of BM-MSCs-derived exosomes.BM-MSCs-derived exosomes of MM patients with EMD increased m RNAs expression of EMT(Vimentin,Twist)and stemness(NANOG,OCT-4)-related markers of U266,LP-1 cell lines;increased proteins expression of EMT-related markers(N-Cadherin,Vimentin,?-Catenin),decreased proteins expression of E-Cadherin of U266,LP-1 cell lines and primary myeloma cells.In addition,BM-MSCs-derived exosomes of MM patients with EMD enhanced invasion of U266,LP-1 cell lines and primary myeloma cells.The mi R-199 a and mi R-197 are increased in the BM-MSCs-derived exosomes of MM with EMD compared with that without.In conclusion,this study demonstrated that BM-MSCs-derived exosomes of MM patients with EMD could be taken up by myeloma cells and these exosomes could induct epithelial-mesenchymal transition(EMT)-like phenotype,cancer stem cell phenotype and enhanced invasion of myeloma cells.We are aimed to further elucidate the mechanism of the occurrence of EMD and provide new targets for the treatment of myeloma patients with EMD.