| Background and Objective: MLL-AF9 acute myeloid leukemia has a high degree of malignancy,poor chemotherapy effect,poor prognosis,and has a unique gene expression profile.To elucidate the pathogenesis of MLL-AF9 acute myeloid leukemia and to find specific and effective targeted therapy is the focus of current research.ADP ribosylation factor 6(ARF6)is a low molecular weight GTP binding protein that plays an important role in actin cytoskeleton formation and cell membrane endocytosis in non-hematopoietic cells.Its role in hematopoietic stem cells remains unclear,so this study initially discussed the regulation of hematopoietic stem cells(HSCs)and the effect of ARF6 on the biological function of AML cells induced by MLL-AF9.Methods: 1.The mutant activated ARF6 mouse model(ARF6-Q67L)was constructed and the ratio of LSK in bone marrow and spleen of wild type(WT)mice and ARF6-Q67 L mice was analyzed by flow cytometry.2.The hematopoietic stem cells of ARF6-Q67 L mice were infected with MLL-AF9(MA)retrovirus to establish a MLL-AF9 induced ARF6-Q67 L mouse AML cell line.3.Bone marrow transplantation experiment was used to observe the effect of ARF6 activated MLL-AF9 leukemia cells on the survival time of mice.4.The effects of ARF6 activation on the biological function of MLL-AF9 AML cells were investigated by cloning formation assay,Giemsa staining,Transwell assay and Annexin ? assay in vitro.5.The effect of activation of ARF6 on signal transduction of ERK and AKT was detected by Western Blot.The expression level of p19 mRNA was detected by RT-PCR.Results: 1.In steady state,the activation of ARF6 did not affect the ratio of bone marrow HSCs and differentiation cells in mice.Under stress condition,14 days after 5-FU injection,the number of HSCs in bone marrow of ARF6-Q67 L mice was obviously damaged.The number of HSCs in spleen increased significantly.2.After activation of ARF6,the survival time of leukemia mice was shortened.3.The results of CFU showed that the number of clones in MA-ARF6-Q67 L group was less than that in MA-WT control group.4.The results of Giemsa staining showed that there was no nuclear differentiation in the MA-ARF6-Q67 L group compared with the MA-WT control group.5.In vitro migration assay showed that the number of cells migrated in MA-ARF6-Q67 L group was 2.8 times higher than that in MA-WT control group.6.MA-ARF6-Q67 L group showed strong resistance to Ara-c.7.Western Blot assay showed that the phosphorylation levels of ERK and AKT in the MA-ARF6-Q67 L experimental group were significantly higher than those in the control group.8.The results of RT-PCR showed that the mRNA expression of p19 in the MA-ARF6-Q67 L experimental group increased.Conclusions: In summary,this study proposes that ARF6 is involved in the recovery of mouse hematopoietic system after injury,and successfully established MLL-AF9 induced mouse leukemia cell line,which provides a new direction for the future research of targeted therapy for leukemia.The main conclusions are as follows:1.Under homeostasis,ARF6 does not affect the proliferation and differentiation of bone marrow hematopoietic stem cells,but during the repair process of bone marrow injury,ARF6 negatively regulates the proliferation of bone marrow hematopoietic stem cells,and positively regulates the proliferation of spleen hematopoietic stem cells to participate in the recovery of hematopoietic system after bone marrow suppression under stress.2.In vivo experiments confirmed that ARF6 accelerated the development of MLL-AF9 AML,and in vitro experiments confirmed that ARF6 inhibited the clonal formation ability of MLL-AF9 AML cells,and promoted the migration and drug resistance of MLL-AF9 AML cells. |
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