Study The Effects Of Bone Marrow Mesenchymal Stem Cells On Stemness Of Multiple Myeloma Cell Lines And Its Mechanism

Objective:1.To investigate the effects of normal bone marrow mesenchymal stem cells(BM-MSCs)on the stemness of multiple myeloma cell lines(LP-1 and U266)in vitro.2.To investigate the effects of bone marrow mesenchymal stem cells(BM-MSCs)of patients with myeloma on the stemness of multiple myeloma cell lines(LP-1 and U266)in vitro.3.To explore the mechanism of the effect of bone marrow mesenchymal stem cells(BM-MSCs)on the stemness of multiple myeloma cell lines(LP-1 and U266).Methods:1.Isolation,culture of bone marrow mesenchymal stem cells.2.The expression of the key stemness genes(OCT4,SOX-2 and NANOG)in control myeloma cell lines(LP-1 and U266),myeloma cell lines directly contacted with BM-MSCs and co-cultured with BM-MSCs through transwell by reverse transcription-PCR.3.The protein levels(OCT-4A,SOX2 and NANOG)in control group,myeloma cell lines directly contacted with BM-MSCs and co-cultured with BM-MSCs through transwell by Western Blot.4.Soft agar clonogenicity assay was assessed after 21 days of culture,Representative images of colonies are shown using an inverted microscop.5.BTK gene expression in co-culture groups(direct contact group and transwell group)compared to control group.6.The expression of the key stemness genes(OCT4,SOX-2 and NANOG)in myeloma cell lines directly contacted with BM-MSCs and co-cultured with BM-MSCs through transwell after adding BTK inhibitor PCI-32765 by reverse transcription-PCR.7.The protein levels(OCT-4A,SOX2 and NANOG)in myeloma cell lines directly contacted with BM-MSCs and co-cultured with BM-MSCs through transwell after adding BTK inhibitor PCI-32765 by Western Blot.8.The myeloma lines LP-1 and U266 co-cultured with BM-MSCs in the absence and presence of PCI-32765 for 72 h and soft agar clonogenicity assay was assessed after 21 days using an inverted microscope.Results:1.The increased expression of key stemness genes(OCT4,SOX-2 and NANOG)in myeloma cell lines was directly contacted with BM-MSCs and co-cultured with BM-MSCs through transwell.2.The increased expression of key stemness proteins(OCT4A,SOX-2 and NANOG)in myeloma cell lines was directly contacted with BM-MSCs and co-cultured with BM-MSCs through transwell.3.LP-1 and U266 cell lines in co-culture(direct contact with BM-MSCs and transwell)groups had a better performance in clonogenicity formation capabilities than control group.4.BM-MSCs promoted BTK gene expression in co-culture groups compared to control group.5.RT-PCR showed that the key stemness genes OCT4,SOX-2 and NANOG decreased in m RNA levels with inhibitors(PCI-32765)in co-culture group.6.Western bolt showed that the key stemness protein(OCT-4A,SOX2 and NANOG)levels decreased with inhibitors(PCI-32765)in co-culture groups.7.Soft agar cloning ability decreased after LP-1 and U266 cells co-culture of BM-MSCs in the presence of PCI-32765.Conclusion:LP-1 and U266 cells were co-cultured with normal BM-MSCs and BM-MSCs of MM patients,the increased expression of stemness genes and proteins could be observed.Therefore,this study showed that BM-MSCs could promote the stemness of multiple myeloma cell lines by directly contacted with multiple myeloma cells and cytokine secretion.A small molecule compounds,BTK inhibitor,PCI-32765decreased the expression of stemness genes and proteins in co-culture group,and inhibited colony-formationability of LP-1 and U266 cells.The results indicated that BTK inhibitor PCI-32765 inhibited the increase of BM-MSCs in the multiple myeloma cell lines.In conclusion,this study demonstrated that BM-MSCs,as a new key player,can promote the MM stemness.Targeting BTK with PCI-32765 can suppress MMSCs and cure myeloma in clinics.But this study has some limitations,the mechanisms BM-MSCs promoting stemness of myeloma cells need further verification.