| IntroductionChronic myeloid leukemia (CML) is a clonal myeloproliferative disorder disease thatoriginates from leukemia stem cells (LSCs) and is not curative. It is widely believed thatLSCs is transformed by BCR-ABL ocogene, a constitutively active tyrosine kinase, andhave a self-renewal and proliferation advantage over normal HSCs to drive expansion ofthe LSCs and is therefore considered causative in the disease. Targeting BCR-ABL toeliminate LSCs is a promising strategy for curing CML. Gleevec (Imatinib Mesylate，IM), atargeted competitive inhibitor of BCR-ABL tyrosine kinase, efficiently induces rapidcytogenetic responses in the majority of patients and has emerged as the first-line treatmentfor CML patients. However, it has been demonstrated that IM predominantly targetsdividing mature CML cells but not LSCs. Therefore, IM fails to eliminate completely LSCsin CML patients and thus is not curative. Elucidation of the mechanism by which LSCsresistance to IM-induced apoptosis is critical for targeting LSCs to eliminate CML.Numerous studies have show that deregulation of miR-21is closely related to drugresistance of cancer cells, and inhibition of miR-21can effectively increase the apoptosisinduced by chemotherapeutic drugs in cancer cells. The expression of miR-21and itsrelationship with resistance to IM-induced apoptosis in CML LSCs is not known so far andrequire further evaluation.In this study, Real-time PCR was employed to detect expression of miR-21in bone marrow mononuclear cells and CD34+stem/progenitor cells from CML patients and healthydonors. On this basis, whether and how miR-21regulates effectiveness of imatinib therapyin CML CD34+stem/progenitor cells were investigated through flow cytometry, agomiR-21or antagomiR-21transfection and so on.ObjectiveThe objectives of the present study are to investigate whether and how miR-21regulates effectiveness of imatinib therapy in CML stem/progenitor cells, providingscientific evidence and reference for cure CML through targeting LSCs.Methods1. To investigate whether miR-21regulates effectiveness of imatinib therapy in CMLstem/progenitor cells.1.1Mononuclear cells were enriched by Ficoll density gradient centrifugation. CD34+stem/progenitor cells were then isolated using a positive magnetic bead selection protocoland molecular phenotype of CD34cells was identified by flow cytometry.1.2CML CD34+stem/progenitor cells treated with IM were stained with Annexin V/PI,and then cell apoptosis were measured by flow cytometry. qRT-PCR was used todetected the expression of miR-21in CML CD34+stem/progenitor cells treated withIM. And flow cytometry was employed to detect the apoptosis induced by IM in CMLCD34+stem/progenitor cells transfected with AntagomiR-21.1.3MTS assay was employed to investigate the effects of IM on the proliferation of Ph+leukemia Sup-b15cells. The apoptosis induced by IM was observed through flowcytometry. qRT-PCR was used to detected the expression of miR-21in Ph+leukemiaSup-b15cells treated with IM. Sup-b15cells were transfected with antagomiR-21, andthe apoptosis induced by IM was measured by flow cytometry.1.4MTS assay was employed to investigate the effects of IM on the proliferation of Ph+leukemia K562and Ku812cells. The apoptosis induced by IM was observed throughflow cytometry. qRT-PCR was used to detected the expression of miR-21in Ph+leukemia K562and Ku812cells treated with IM. K562and Ku812cells were transfected with agomiR-21, and the apoptosis induced by IM was measured by flowcytometry.2. To identify the molecular mechanism of miR-21-mediated resistance of CMLCD34+cells to IM-induced apoptosis2.1Flow cytometry was used to detected the PTEN and PDCD4protein levels in CMLCD34+stem/progenitor cells, Sup-b15, K562and Ku812cells were treated with IM,and the PTEN、PDCD4and p-AKT protein levels in CML CD34+stem/progenitor cellstransfected with AntagomiR-21.2.2CML CD34+stem/progenitor cells and Sup-b15cells were treated with PI3K inhibitorand IM together, and then the cell apoptosis was detected through flow cytometry.2.3CML CD34+stem/progenitor cells and Sup-b15cells were treated with PI3K inhibitorand IM together, and then the p-AKT protein level was detected by flow cytometry.Result1. To investigate whether miR-21regulates effectiveness of imatinib therapy in CMLCD34+stem/progenitor cells.1.1The flow cytometry result showed that the purity of CD34+stem/progenitor cellspurified with magnetic activated cell sorting system is89.23%.1.2We observed only a small apoptosis was seen in CML CD34+stem/progenitor cellsafter IM treatment, and miR-21expression slightly increased in CML CD34+stem/progenitor cells exposed to IM. In addition, transfecting with antagomiR-21notably increased apoptosis in IM-treated CML CD34+stem/progenitor cells comparedto the cells transfected with antagomiR-NC, the apoptosis rate increased from3.983±0.515%to9.437±0.970%.1.3Sup-b15cells exhibited resistance to IM-induced apoptosis. And the miR-21expressionwas slightly increased in Sup-b15cells exposed to IM. Transfecting with antagomiR-21also markedly sensitized Sup-b15cells to IM-induced apoptosis.1.4IM significantly decreased cell viability and induced apoptosis in K562and Ku812cellsin a time-and concentration-dependent manner. The expression of miR-21wasmarkedly reduced in K562and Ku812cells after treated with IM. Transfecting with agomiR-21significantly and modestly inhibited imatinib-induced apoptosis in Ku812and K562cells, respectively.2. To identify the molecular mechanism of miR-21-mediated resistance of CMLCD34+stem/progenitor cells to IM-induced apoptosis.2.1The treatment of IM resulted in a reduction in PDCD4and PTEN protein levels in CMLCD34+stem/progenitor cells. IM also decreased PDCD4and PTEN expression inSup-b15cells, but increased the PDCD4expression and reduced PTEN expression inK562and Ku812cells.The inhibition of miR-21by antagomiR-21decreaed thePDCD4、PTEN and p-AKT expression in CML CD34+stem/progenitor cells.2.2Combination of PI3K inhibitor with IM dramatically increased the apoptosis induced byIM in CML CD34+stem/progenitor cells and Sup-b15.2.3Combination of PI3K inhibitor with IM dramatically decreased p-AKT in CML CD34+stem/progenitor cells and Sup-b15.ConclusionMiR-21modulates apoptosis resistance of CML CD34+stem/progenitor and Sup-b15cells to IM through PI3K/AKT pathway. |
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