A Preliminary Study Of The Role Of ARF6 On Regulation Of Hematopoiesis In Mice And Promoting The Proliferation Of Chronic Myeloid Leukemia Cells Induced By BCR-ABL

A preliminary study of the role of ARF6 on regulation of hematopoiesis in mice and promoting the proliferation of chronic myeloid leukemia cells induced by BCR-ABLBackground and purpose:Chronic myeloid leukemia（CML）is a malignant tumor of the blood system,mainly due to the loss of proliferation,differentiation disorder and obstruction of apoptosis of leukemia cells.95%of CML patients are induced by the BCR-ABL fusion gene,which is produced by chromosome translocation of 9 and 22 and encodes a 210k Da continuously activated tyrosine kinase,which can induce the occurrence of CML.The current treatment mainly focusses on tyrosine kinase inhibitors such as Imatinib,which target BCR-ABL,which have achieved good clinical results.But this targeted drug cannot completely cure CML,and long-term use of Imatinib is prone to drug resistance.Clarifying the molecular mechanism of CML cell proliferation and finding a new treatment target for CML are the future research directions and focusses of CML diseases.ADP-ribosylation factors（ARFs）are widely expressed and highly conservative GTP binding proteins in eukaryotic cells and are a member of the Ras superfamily.ARF6,as one of the special subtypes,mainly participates in the transportation of cell membranes and transmembrane protein substances.ARF6 has also been found in recent years to promote the infiltration,migration and proliferation of glioma cells in breast cancer and melanoma cells.ARF6’s research on tumors has always focused on solid tumors and played the role of oncogenes,rarely reported in leukemia.Until 2013,Sunsuke and other studies found that after the GAP protein Smap of ARF6 was knocked out,mice developed bone marrow hyperplasia syndrome（MDS）and acute myeloid leukemia（AML）phenotypes,suggesting that the continuous activation of ARF6 may promote the occurrence of MDS and AML.In order to determine whether ARF6 plays the same biological function in CML cells,we constructed the BCR-ABL（BA）-induced preleukemia cell line of ARF6,and used this model to study the biological function and mechanism of ARF6 in CML cells.At the same time,we also used hematopoietic cell-specific knocking on mice and the effect of ARF6 deletion on hematopoietic stem/progenitor cells function and spectral differentiation in mice,so as to clarify the hematopoietic regulatory function of Arf6 and evaluate whether it can become a target of CML treatment.Method:1.Construct two Arf6 knockout transgenic mice of Cre-LoxP system,i Vav Cre Arf6^loxp/loxpand Mx1Cre⁺Arf6^loxp/loxprespectively,for in vivo and in vitro experiments.Use PCR method to identify the mouse genotype;2.Study the important role of Arf6 in the hematopoietic system by analyzing the changes the total number of hematopoietic stem cells/progenitor cells in the bone marrow and spleen and the proportion of other hematopoietic mature cells in the mouse model of induced knockout of Arf6 by flow cytometry analysis to study the important role of Arf6 in the hematopoietic system;3.Making the retrovirus of BCR-ABL and use it to construct a preleukemia cell line of chronic myeloid leukemia induced by BCR-ABL by knocking out Arf6;4.Through in vitro growth curve,clone formation experiments and Giemsa experiments,specifically detect the biological function of Arf6 in chronic myeloid leukemia;5.Through qPCR,Western Blot and other molecular biology experiments to study the possible regulatory signal pathways that Arf6 plays a role.Result:1.The results of flow cytometry showed that the total number of hematopoietic stem cells and hematopoietic progenitor cells in the bone marrow and spleen of Arf6knockout mice was not significantly different from that of the control group（P>0.05）;2.Hematopoietic analysis found that compared with the control group,the percentage of B cells in the bone marrow and spleen of the mice that knocked out Arf6decreased,the percentage of megakaryocytes increased,and the percentage of T cells in the bone marrow increased.the proportion of myeloid cells in the spleen increased while the proportion of red blood cells decreased.;3.The qPCR results showed that after knocking out Arf6,shows that Arf6knockout does not affect the expression of other genes in the family,and also implies they did not play a compensatory role;among the genes related to the cell cycle,the expression of p21 and p27 increased;4.Knockout of Arf6 does not affect the cloning ability of primary bone marrow cells;5.In vitro growth curve display the proliferation of leukemia cells without Arf6was 50%less that of the experimental group;6.The experimental results of cell clone formation showed that the number of pre-leukemia cell clones that knocked out Arf6 was reduced by 33%compared with the control group;7.The results of Giemsa staining showed that knocking out Arf6 doesn’t affect the differentiation of preleukemia cells;8.Western Blot results showed that Arf6 knockout greatly reduced the phosphorylation levels of AKT and ERK in pre-BA leukemia cells,and increased the protein expression of p21 and p27 at the same time.Conclusion:1.Arf6 deletion does not affect the proliferation and differentiation of mouse hematopoietic stem/progenitor cells under normal conditions,but which affects the differentiation of mature hematopoietic cells in bone marrow and spleen;2.After Arf6 is knocked out in mice,other genes in the Arf family will not have a compensatory effect;3.Knockout of Arf6 inhibited the proliferation of CML preleukemia cells induced by BCR-ABL;4.Arf6 may promote CML cell proliferation by regulating PI3K/AKT and ERK signaling pathways and inhibiting the expression of p21 and p27 related cell cycle genes.