Study On Function Of ARF6 In MLL-AF9-induced Acute Myeloid Leukemia Cells

Background and objective:Acute myeloid leukemia??AML??is a kind of hematological myeloid malignant tumor with high clinical heterogeneity,which is characterized by uncontrolled proliferation and disturbance of differentiation and maturation of hematopoietic stem/progenitor cells in bone marrow.2-5%of them are acute myeloid leukemia induced by MLL-AF9.Clinical chemotherapy and bone marrow transplantation rarely completely cure acute myeloid leukemia,and most AML patients will develop drug resistance and recurrence.The root cause is that leukemia stem cells with self-renewal and chemotherapy resistance cannot be eradicated.At present,the gene diagnosis and targeted drug research around the individualized targeted therapy of AML are the focus and focus of clinical treatment of AML,including cytotoxic chemotherapy reagents,monoclonal antibodies and targeted small molecule inhibitors.ADP ribosylation factor 6?ARF6?is a GTP binding protein that participates in the transport of cell biofilm and transmembrane proteins.ARF6 has been proved to play an important role in promoting the proliferation,invasion and migration of many kinds of tumor cells.The purpose of this study is to explore the specific role of ARF6 in the occurrence and development of acute myeloid leukemia induced by MLL-AF9,which is of great significance to clarify the role and mechanism of ARF6 in regulating the occurrence and development of AML.Methods:1.Arf6^f/f transgenic mice based on Cre-LoxP system were constructed and genotypes were identified by PCR.2.MLL-AF9,Migr1 and Cre retroviruses were coated to construct ARF6 knockout MLL-AF9-induced acute myeloid leukemia cell lines.3.It was determined that ARF6 was involved in the specific function of MLL-AF9-LC through cell functional experiments,such as growth curve test,clone formation test,migration test and drug resistance test.4.It was determined the effects of knockout ARF6 on other possible signaling pathways through molecular biology experiments,such as Western blot,PCR,RT-PCR,qPCR,etc.5.Through the experiment of bone marrow transplantation,to explore the effect of ARF6 knockout cells on the survival time of mice.6.Construction of hematopoietic knockout ARF6mouse model Vav-iCre⁺,Arf6^f/f.The changes of the proportion of cells in bone marrow and spleen of Vav-iCre⁺,Arf6^f/f mice were observed by flow staining analysis to explore the role of ARF6 in normal hematopoietic cells.Results:1.The results of cell growth curve in vitro showed that the number of cells knocked out of ARF6 in MA-Cre was 2.6 times less than that in MA-Migr1?P<0.0001?.2.The results of cell colony formation assay showed that the total number of clone formation of MA-Migr1 was 1.4 times higher than that of MA-Cre?P<0.0001?.3.The results of cell migration assay showed that the number of migrated cells in MA-Migr1 was 1.8 times higher than that of MA-Cre?P<0.0001?.4.The results of apoptosis showed that the apoptosis rate of MA-Cre was 1.1 times higher than that of MA-Migr1?P<0.001?.5.The results of bone marrow transplantation in mice showed that the survival time of mice transplanted with MA-Cre was 2.6 times longer than that of mice transplanted with MA-Migr1?P<0.001?.6.The results of Western blot assay showed that knockout of ARF6 greatly reduced the phosphorylation level of AKT in MA cells.7.The results of flow analysis showed that there was no significant difference in the proportion of hematopoietic cells in bone marrow and spleen between Vav-iCre⁺,Arf6^f/f mice with complete knockout of ARF6 and the control group.8.The results of proliferation and cloning experiment in vitro showed that complete knockout of ARF6 greatly damaged the proliferation and cloning ability of MA-pre-LC cells.9.The results of flow cytometry showed that complete knockout of ARF6 did not affect the level of apoptosis of MA-pre-LC cells.Conclusion:1.Knockout of ARF6 does not affect the hematopoietic function of normal mice.2.Knockout of ARF6 inhibited the proliferation and migration of MA acute myeloid leukemia cells.3.Knockout of ARF6 prolonged the survival time of mice with MA.4.ARF6 participates in the regulation of PI3K/AKT signaling pathway.