The Role And Mechanism Of MIF In The Crosstalk Between Leukemia Cells And MSCs

Backgrounds:Leukemia is a highly heterogeneous disease with worse prognosis characterized by malignant clonal proliferation of hematopoietic stem/progenitor cells.Currently a higher complete response(CR)rate was observed in leukemia,but the 5 years survival rate was only about 25%.Thus,it is important to explore the pathogenesis of leukemia.Macrophage migration inhibitory factor(MIF)is an important multifunctional cytokine and overexpressed in leukemia as well as many solid tumors including pancreatic cancer,breast,prostate and colon cancers,which associated with poor prognosis.Bone marrow microenvironment(BMM)creates a unique niche to facilitate the malignant behaviors of leukemia cells involving in invasion,metastasis,drug resistance and recurrence,etc.Mesenchymal stem cells(MSCs)are one crucial member of BMM.However,the role of the crosstalk between MSCs and leukemia cells is still unclear.The project in vitro investigate the expression level of MIF in patients with leukemia,and IL-8 as well as NF-κB/p65 were up-regulated in the bone marrow derived MSCs of leukemia patients,which contribute to promote the invasion capacity of leukemia cells.It elucidates the role of BMM in the pathogenesis of leukemia,and provides evidence for digging the therapy targets of leukemia as well as making for the prevention and control strategies.Aim:To explore the role and mechanism of MIF in the leukemia microenvironment modulated the bone marrow MSCs to promote the invasive ability of leukemia cells.Methods:1.BM-MSCs were cultured in DMEM medium,harvested by digesting using trypsin,which were identified by induce to differentiate into fat-like cells,osteoblast-like cells and chondrocyte-like cells.2.Application of ELISA for the determination of MIF in the bone marrow fluid,plasma from 50 AML patients and the control group.The expression of IL-8 was detected in the co-culture supernatant of leukemia cells and BM-MSCs as well as their cultured alone.3.Cell proliferation was assessed by cell counting kit-8(CCK-8)in leukemia cells co-cultured with BM-MSCs as well as their cultured alone.4.Quantitative Real Time PCR analysis(qPCR)was used to determine IL-8mRNA expression in BM-MSCs after incubated with rhMIF or co-cultured,cultured alone leukemia cells.5.The expression of NF-κB/p65 was examined by western blot in BM-MSCs after incubated with rhMIF or exosomes derived from the supernatant of culture K562 cells or co-cultured with leukemia cells.6.The invasion model of Trans-Well system containing Matrigel was used to detect the effect of MIF inhibitor and a specific inhibitor of NF-κB signal pathway on the invasion capability of K562 cells.7.Exosomes derived from culture supernatant of K562 as well as the peripheral blood were separated using Exo Quick Precipitation of SBI kit.MIF mRNA expression carried in exosomes was chosen to be validated by quantitative RT-qPCR Western Blot test was used to examine the effect of exosomes on the expression of p65 in the BM-MSCs.Results:1.BM-MSCs grew adherently to the wall showing long fusiform with spindle cell morphology with the potential to differentiate into fat-like cells,osteoblast-like cells and chondrocyte-like cells.2.The ELISA showed that the MIF levels in the bone marrow fluid and plasma of AML group were(24.86±7.66)ng/mL and(60.49±12.12)ng/mL,respectively.They are higher than those in the control group(5.31±2.62)ng/mL and(2.05±1.10)ng/mL.There was significantly statistically(P<0.001).3.The counts of leukemia cells after co-culture with BM-MSCs were significantly higher than that in the control group(P<0.001).4.The MIF、 IL-8 levels were increased in the supernatant after co-culture with BM-MSCs compared with the control group,and the difference was significant,respectively(P<0.001).5.The expressions of IL-8 and NF-κB/p65 in BM-MSCs were up-regulated significantly by rhMIF(P<0.001).6.The invasion ability of leukemia cells co-cultured with BM-MSCs was decreased by MIF inhibitor and a specific inhibitor of NF-κB signal pathway,with an inhibitory rate of(45.00±0.17))% and(47.00±0.14)%.Treated by combination ISO-1and BAY-11-7082,the K562 cells invasion rate was(38.00±0.15)%,which there were statistical significance as compared with the control group(P<0.05).7.The peripheral blood derived exosomes carried MIF mRNA,and its expression was up-regulated in patients with leukemia.K562 exosomes can promote the expression of NF-κB/p65 in BM-MSCs,which was attenuated by ISO-1 and BAY-11-7082(p<0.05).Conclusion:The BM-MSCs can be effectively cultured and expanded from AML patients,which possess the differentiated capability.The MIF expression level was increased in the bone marrow fluid and plasma from patients with AML.The expression level of IL-8 was up-regulated by MIF through NF-κB/p65 signal pathway in BM-MSCs,and promoted invasion capacity of K562 cells.The peripheral blood derived exosomes loaded with MIF mRNA.The protein expression of p65 in BM-MSCs was increased by exosomes derived from K562 cells,which may be related to exosomes loaded with MIF.