The Studies On Molecular Mechanism Of ARF6in Promoting Human Chronic Myelogenous Leukemia K562Cells Proliferation And Drug Resistance

Chronic myeloid leukemia (CML) is a clonal hematologic malignancy which originatesfrom pluripotent hematopoietic stem cell, and it accounts for15%~20%of adult leukemia.About95%of CML patients were caused by BCR-ABL fusion gene, which results from the9thand22ndchromosome translocation, encoding a constitutively activated tyrosine kinaseto induce the occurrence of CML. For CML therapies, Imatinib targeted BCR-ABL kinasehave achieved better clinical curative effect, and prolonged survival time of CML patientswho could not transplant hematopoietic stem cell. However, Imatinib can not cure CML,because its long-term application easily led to gene mutation of BCR-ABL and enhanceddrug resistance. More importantly, Imatinib cannot eliminate leukemia stem cells (LSCs),and then results in the relapse of disease, so to search the other drug target exceptBCR-ABL kinase will be a key point to cure CML and completely remove LSCs.In recent years，RacGTPs have been hotspot genes involved in the development of CML,and located at the downstream of BCR-ABL through regulating Ras/MAPK, PI3K/Akt,FAK and Stat5signal transduction and Bcl-XL gene expression to promote CML cellproliferation, migration and drug resistance. In the RacGTPs gene family, Rac1wasconsidered as a most important gene relative to CML development. Rac1activationinduced by BCR-ABL enhanced leukemic cell drug resistance and maintained LSCsmicroenvironment. However, the mechanism of Rac1activation in CML is still not clear.Lately, a small GTP binding protein, ARF6, was found by its own activation andinactivation state to regulate Rac1activity. whether ARF6mediated BCR-ABL-Rac1signaling pathway and whether ARF6play a key role in BCR-ABL induced CML, becomethe starting point of this study. Based on these questions, we will apply molecular biology,cell biology and genetics methods to explore the role of ARF6in BCR-ABL induced CMLand the possible molecular mechanism. These studies will be helpful to elucidate thepathogenesis of CML, and find novel targets for clinic and drug development. In order to study the role of ARF6in BCR-ABL induced CML, we established a ARF6knockdown K562cell line (shARF6), compared with control group cell line (ARF6-NC),to study the effects of ARF6on proliferation, survival, differentiation and drug resistanceof K562cells. Meantime, the molecular mechanisms of ARF6in promoting theproliferation and drug resistance of K562cells will be elucidated.Part1: Effects of ARF6on proliferation, survival and differentiation of K562cellsMethods:1.Using CFU to detect the effect of ARF6on colony formation ability of K562cells.2. Using CCK8to detect the effect of ARF6on the proliferation and sensitivity todrugs of K562cells.3. Using Annexin V staining to detect the effect of ARF6on cellsurvival of K562cells.4. Using CD14staining and Giemsa staining to analyze the effect ofARF6on the differentiation of K562cells.Results:1The CFU results showed that the number of clone in K562-shARF6wassignificantly less than that of the control group ARF6-NC.2The CCK8results showed thatcell proliferation rate in K562-shARF6was significantly lower than that of the controlgroup; the K562-shARF6cells were more sensitive to Imatinib and Ara-c compared withthe control group.3Annexin V staining showed that the apoptosis rate of K562-shARF6cells has no significant difference compared with the control group.4CD14stainingshowed that the CD14positive rate of K562-shARF6cells had no obvious differencecompared with K562-NC cells. Further, Giemsa staining results indicated thatK562-shARF6cells had no obvious nucleus differentiation characteristics.Part2: The molecular mechanisms of ARF6promotes K562cell proliferation anddrug resistanceMethods: Western blot was used to detect activation of Stat5, ERK and AKT signaltransduction and expression of cyclinB1, c-myc, p21and A1Results: Compared with control group, downregulaion of ARF6inhibited phosphorylationof Stat5and AKT, but does not affect ERK phosphorylation; ARF6silence upregulates p21gene expression, downregulates the expression of cyclinB1, c-myc and A1.Conclusions:ARF6promotes K562cell proliferation and colony formation ability, and enhances K562 cell resistance to Imatinib and Ara-c, and the preliminary results display ARF6isindispensable for CML proliferation and drug resistance by activating STAT5and AKTsignals and regulating the expression of c-myc, cyclinB1, p21and A1.