The Role Of ZFX In Chronic Myeloid Leukemia Stem/Progenitor Cells

Objective:This thesis aims to delineate the role of Zinc finger protein,X-linked(ZFX)in chronic myeloid leukemia stem/progenitor cells and its underlying mechanisms.Methods:(1)Q-RT-PCR and western blot were untilized to assess the expression of ZFX in primary and CD34+cells derived from healthy donors and CML patients.(2)We silenced ZFX expression and investigated the effects on colony-forming cell(CFC)ability of CD34+cells from CML patients.(3)The CFC detection and Annexin V/7-AAD apoptosis analysis were conducted after IM treatment coordinated with ZFX silencing in CML CD34+cells(IM-resistant defined by in vitro assay)to evaluate its impact on IM sensitivity.(4)Microarray was utilized to analyse the gene expression influenced by ZFX silencing and the potential target gene WNT3 was further validated by Q-RT-PCR,ChIP and luciferase assay.(5)The cell growth,CFC ability and IM reactivity were measured in K562 cells upon WNT3 silencing.(6)The expressions of several molecules in Wnt/β-catenin signaling pathway were measured by Q-RT-PCR and western blot in ZFX-silenced K562 cells.Results:(1)ZFX was expressed aberrantly in CML CD34+cells,and the expression was increased along with the disease development.(2)ZFX silencing had a significant inhibitory effect on colony-forming cell(CFC)ability in CML CD34+including IM-resistant CD34+cells(defined by in vitro assay)and augmented cellular apoptosis induced by IM.(3)It was validated that WNT3 was the target gene of ZFX,and WNT3 expression was increased in CML CD34+cells compared to the control cells.(4)WNT3 silencing declined the cell growth and CFC ability and enhanced IM sensitivity of K562 cells.(5)WNT3 overexpression partically "rescued" the growth inhibition and IM hypersensitivity caused by ZFX silencing.(6)Targeting ZFX downregulated the expression of several molecules in Wnt/β-catenin signaling pathway.Conclusion:ZFX is expressed aberrantly in CML CD34+cells.ZFX silencing inhibits the growth and IM resistance of CML cells with increased apoptosis.ZFX modulates the transcription of WNT3 by binding its promoter.ZFX regulates the growth and IM reactivity of CML cells partially via Wnt/β-catenin signaling pathway.