Effects Of Downregulated ARF6 On Biological Characteristics Of MLL-AF9-induced Acute Myeloid Leukemia Cells

Background and objective:As a highly heterogeneous malignancy,acute myeloid leukemia（AML）is char-acterized by impaired differentiation and uncontrolled proliferation of myeloid pre-cursor clones.The 5-year relative survival rate for AML is only 27.4%,with relapse occurring in 40%-50%of patients.Approximately 10%of AML patients have an MLL fusion gene,with MLL-AF9 fusion leukemia occurring in up to 34%of cases.Until now,there are no targeted drugs for MLL-AF9-induced AML,and relapse after conventional chemotherapy and bone marrow transplantation is very common,and few patients are completely cured.Targeted therapy has been a hot topic and direction of research in AML,however,we still know little about the pathogenesis of AML and potential therapeutic targets,therefore,the elucidation of the pathogenesis of AML and the discovery of new drug targets are important for the future treatment of AML.ARF6 is a member of the ADP-ribosylation factor（ARF）family with a molecu-lar weight of approximately 20 k D and is localized to the plasma membrane,cyto-plasm and endosomal membrane.ARF6 has been shown to regulate a variety of cellu-lar functions such as actin cytoskeleton remodeling,extracellular secretion,cytosolic recycling,cell division,membrane lipid metabolism,microcyst transport and release,etc.ARF6 has been shown to have key functions in tumor proliferation,migration and invasion.The group discovered that ARF6 has the biological function of promoting the proliferation of chronic myeloid leukemia cells and reducing their sensitivity to chemotherapeutic drugs.In order to clarify whether ARF6 has similar biological ef-fects in AML,this study used ARF6 knockout mouse AML cells and Arf6 down-regulated human THP-1 cells as the subjects to elucidate the biological functions of ARF6 in AML with MLL-AF9（hereinafter referred to as MA-AML）using molecular biology and cell biology approaches,and ultimately provide new ideas and candidate targets for drug development and clinical treatment targeting AML.Methods:1.Bone marrow and spleen cells from Vav-i Cre⁺Arf6^loxp/loxp experimental mice which induced to knock out Arf6 by the Vav-icre-Lox P knockout system and wild type（WT）control mice were extracted by flow cytometric analysis to detect the func-tional effects of in vivo knockdown of Arf6 gene on hematopoietic stem/progenitor cells（HSPCs）in the bone marrow and spleen of mice.To preliminarily evaluate the function of ARF6 in the hematopoietic system of mice.2.MLL-AF9 retrovirus was encapsulated,fluorescence microscopy was per-formed and transfection efficiency was measured for cellular infiltration of mouse CD117⁺cells to construct an MLL-AF9-induced mouse acute myeloid promyelocytic leukemia cell line（MA-pre-LC）,referred to as the MA cell line.3.Construction of two types of cell lines by cell infiltration and flow sorting:（i）mouse MA cell lines,including the experimental MA-Vav-icre⁺-pre-LC cell line with Arf6 knocked out,and the control MA-WT-pre-LC cell line.（ii）human acute mono-nuclear leukemia THP-1 cell line（with MLL-AF9 fusion gene）,including ARF6-sh RNA cell lines with knockdown of Arf6 induced by sh RNA lentivirus,and control NC-sh RNA cell lines;Green fluorescent protein（GFP⁺）cells were screened by flow sorting technique,and the cell lines were identified by q PCR to detect Arf6 knock-down in the experimental groups.4.The function of Arf6 in MLL-AF9-induced acute myeloid leukemia cells was determined by a series of functional assays,such as proliferation assay,clone for-mation assay,cell cycle assay,cell migration assay,apoptosis resistance assay and Giemsa staining.5.Molecular biology methods such as Western blot,PCR and q PCR were used to detect the protein expression levels of related genes and protein kinase B（AKT）and extracellular signal-regulated kinase（ERK）,to explore the involvement of ARF6 in signalling pathways,or regulated gene expression.Results:1.Flow cytometric analysis showed that the hematopoietic cell-specific knock-down of the Arf6 gene may have affected the migration of HSPCs from the spleen to the bone marrow.Compared to control WT mice,the percentage of T cells in the bone marrow of Arf6 knockout mice was significantly higher and the percentage of B cells in the spleen was decreased.2.Fluorescence microscopy results of MLL-AF9 virus envelope showed that the green fluorescence intensity of the cells was high and the GFP⁺efficiency was over80%.3.The results of flow cytometric sorting cell lines showed that:（i）the mouse MA cell line was 100%pure.（ii）the THP-1 cell line was 99%pure.q PCR results showed that the expression of Arf6 gene was significantly inhibited in the experimental group.4.Cell function experiments.（1）The results of the in vitro cell growth curve assay showed that the number of MA-Vav-icre⁺-pre-LC cells was significantly lower com-pared to the control group,only 38.5%of the control group MA-WT-pre-LC;the number of ARF6-sh RNA in THP-1 cells with knockdown of Arf6 was also signifi-cantly lower,66.7%of the control group NC-sh RNA.（2）The results of the cell clone formation experiments showed that the knockdown of Arf6 gene reduced the clono-genic ability of MA cells,with the number of first-generation clones in the MA-Vav-icre⁺-pre-LC group being 52.6%of that in the control group and the number of sec-ond-generation clones in the MA-Vav-icre⁺-pre-LC group being only 45%of that in the control group.Similarly,knockdown of the Arf6 gene reduced the clonogenic abil-ity of THP-1 cells,with the number of first-generation clones in the ARF6-sh RNA group being 71.4%of the control NC-sh RNA and the number of second-generation clones in the ARF6-sh RNA group being only 59%of the control NC-sh RNA.（3）The cell cycle assay showed that downregulated of the Arf6 gene affected the cell cycle of MA cells or THP-1 cells,the percentage of S-phase,MA-Vav-icre⁺-pre-LC was only12.8%of the control MA-WT-pre-LC,20.8%of the ARF6-sh RNA group compared to the NC-sh RNA group;the percentage of G₂/M-phase,MA-Vav-icre⁺-pre LC group was 3.9 times higher than that of the MA-WT-pre-LC group,and the ARF6-sh RNA group was 2.7 times compared to the NC-sh RNA group.The percentage of G₀/G₁phase ARF6-sh RNA group was 1.2 times higher than the NC-sh RNA group.（4）The results of the cell migration assay showed that the number of migrated cells in the MA-Vav-icre⁺-pre-LC group was 36%of the control MA-WT-pre-LC group com-pared to the control group;The number of migrated cells in the ARF6-sh RNA group was only 34%of that in the control NC-sh RNA group.（5）The results of the apoptosis assay showed:there was no difference in the proportion of apoptosis between the DMSO-treated Arf6 knockout group and the control group（P>0.05）,the percentage of apoptosis was significantly higher in MA-Vav-icre⁺-pre-LC compared with the MA-WT-pre-LC group,and the percentage of apoptosis in MA-Vav-icre⁺-pre-LC was8.4 times higher at a concentration of 0.6μM of agranulocyte Ara-c,the proportion of apoptosis in MA-Vav-icre⁺-pre-LC was 4.3 folds higher than that in the control MA-WT-pre-LC group at an Ara-c concentration of 1μM.The results of the THP-1 cell line assay showed that the percentage of apoptosis of ARF6-sh RNA was 1.6 times higher than that of NC-sh RNA at Ara-c concentration of 0.6μM.The apoptotic ratio of ARF6-sh RNA at 1μM Ara-c concentration was 1.8 times higher than that of NC-sh RNA.（6）The results of Gimsa staining showed no difference in cell morphology and nuclear morphology between the experimental and control groups for both cell lines.5.Western blot assay results showed that ARF6 low expression has reduced the phosphorylation levels of AKT and ERK in MA-AML cells,the q PCR assay showed that the expression of cell cycle-related genes p21,p16 and p27 was significantly in-creased,the expression of CCND3 was decreased and the expression of transcription-related Stat5 gene was decreased in the MA-Vav-icre⁺-pre-LC and ARF6-sh RNA cell lines.Conclusion:1.Successful construction of MLL-AF9-induced acute myeloid promyelocytic leukaemia cell line.2.Hematopoietic cell-specific knockdown of Arf6 affected the migration of HSPCs between the bone marrow and spleen.3.Knockdown of Arf6 inhibited MLL-AF9-induced proliferation and migration of pre-AML leukemia cells.4.ARF6 downregulation did not affect cell survival,enhanced the sensitivity of MA-AML cells to the chemotherapeutic drug Ara-c without affecting cell differentia-tion.5.ARF6 downregulation may inhibit MA-AML cell proliferation by attenuating PI3K/AKT and ERK signaling pathways and regulating cell cycle-related genes.