The RalA GTpase Activity In Malignant Transformation Of Chronic Myelogenous Leukemia (CML) Cells And Intervention Effects Of MiR-181a

Objective：Our goal is to investigate the potential roles of RalA GTPase activity in malignanttransformation of CML cells and interference effects of miR-181a. This study will be alsoexpected to provide sufficient experimental evidence for molecular therapeutics inleukemia.Methods:1) RalA plasmid （EX-B0058-M03) and empty plasmid （EX-NEG-M03) weretransfected into K562cells through Effectene Transfection Reagent, sorted by G418and FACS.2) The RalA GTPase activity was tested in CML cell lines (K562, KCL-22, Jurl-MK1)and normal blood samples. also, K562cells transfected with RalA plasmid、miR-181a and RalA siRNA were detected through G-LISATMRal Activation AssayBiochem KitTM.3) The location of RalA in K562cell was detected with confocal laser scanningmicroscope.4) Transfecting miR-181a and its candidate target gene RalA siRNA into K562cell byLipofectamineTM2000.5) The relative expression of RalA protein was detected after transfection with RalAplasmid by Western Blot.6) The effects of RalA plasmid、miR-181a and RalA siRNA on K562migration、 invasion activity were tested by Transwell assay.7) The effects of RalA plasmid, miR-181a and RalA siRNA on TKIs sensitivity inK562cells were determined by MTT assay.8) The effects of RalA plasmid、miR-181a and RalA siRNA on K562colony-formingactivity were tested by semi solid culture medium of Methylcellulose.9) Phosphorylated proteins were detected with Protein microarray technology andKEGG pathways were analysed.Results:1) The GFP rate of K562transfection with RalA plasmid was2.60%and92.59%aftersorting with G418and FACS, respectively.2) Compared with normal blood control, the activity of RalA GTPase in CML cells washiher. RalA GTPase activity in RalA Plasmid group promted significantly.and K562cells transfected with miR-181a and RalA siRNA had lower activity of RalA GTPasethan SCR group.3) RalA was mainly localized at cytoplasm of K562cell.4) The relative expression of RalA protein was higher after transfection with RalAplasmid by Western Blot.5) RalA plasmid significantly promoted K562cell migration、invasion and colony-forming ability（P<0.05）.6) The overexpression of RalA significantly resisted to Imatinib and promoted colony-forming ability of K562cells（P<0.05）.7) The sensitivity to Imatinib and Nilotinib was promoted greatly after transfection withmiR-181a and RalA siRNA in K562cells.and the ability of K562cell migration、invasion and colony-forming was suppressed effectively（P<0.05）.8) After transfection with miR-181a and RalA siRNA, the number of phosphorylatedproteins decreased to20%or higher in K562cells is25and11, respectively. theoverlapped number is8.KEGG analysis showed that multiple pathways were related to cancer.Conclusions:1) In CML cells, RalA GTPase activity is high in common.Overexpression of RalA canpromot the malignant transformation of CML K562cell.Targeted inhibition of RalAGTPase activity can surpress K562cell malignant behavior effectively, whichindicate RalA may be a new target for CML.2) Targeted inhibition of RalA significantly downregulated proteins phosphalation levelin multiple Ras dowmstream pathways.