The Research Of Heme Oxygenase-1 In The Myelogenous Leukemia

Objective 1.To explore the relationship between HO-1 expression in PB and progression/relapse of CML,and evaluate to find out a new molecular marker for CML progression.2.Study the effects of heme oxygenase-1 silencing on the survival of AML-M2 cells in vivo and in vitro.3.Aimed to explore the biological behaviors and mechanisms?e.g.AML cell apoptosis and drug resistance?of silenced HO-1 in Kasumi-1 cells.Methods 1.A total of 60 peripheral blood and bone marrow samples from 25 CML patients in different phases were collected to detect the expressions of HO-1 and bcr/abl using real-time PCR.Routine blood test was performed to detect the changes of leukocyte and platelet counts.The proportion of primitive cells in bone marrow was detected by flow cytometry.The relationship between high HO-1 expression and CML progression and relapse was explored by analysis of variance and linear regression analysis.The prediction accuracy and diagnostic cutoff values were determined by receiver operating characteristic curve.2.Gradient centrifugation for separation of bone marrow mononuclear cells by Ficoll was performed.A recombinant lentivirus targeting HO-1 gene,pRNAi-siHO-1-GFP,was constructed to infect BMMNCs.The transfection efficiency was observed by fluorescence microscopy.The influences of daunorubicin on the growth inhibition rate of BMMNCs were detected by Cell Count Kit-8.The apoptotic rate was detected by flow cytometry.The mRNA and proteins expressions of HO-1 and apoptosis-related caspase3,caspase8 and caspase9 were detected by real-time PCR and Western blot respectively.An AML1/ETO-positive Kasumi-1 cell-inoculated AML-M2 xenograft mouse model was established.The tumor formation outcomes were observed,and survival curves were plotted.The counts of leukocytes and platelets and hemoglobin levels were monitored at regular intervals.The copy numbers of AML1/ETO fusion gene of bone marrow,liver,spleen,lung and renal were detected to assess the infiltration condition of leukemic cells.Besides,pathological test:we acquired the mice tissues of liver,spleen,renal and lung and observed the infiltration of inflammatory cells.3,Small interfering RNA with lentivirus was employed to allow targeted silencing of HO-1 in Kasumi-1 cells?AML-M2 cell lines?,The transfection efficiency was observed by fluorescence microscopy and flow cytometry.The influences of daunorubicin on the growth inhibition rate of cells were detected by MTT.Annexin-V-7-AAD method was used to detect cell apoptosis by flow cytometry.The mRNA and proteins expressions of HO-1 and apoptosis-related genes were detected by real-time PCR and western blot respectively.Fluo3-AM probe was used to measure Ca²⁺ accumulation by flow cytometry.DCFH-DA probe was used to measure ROS generation by flow cytometry.JC-1 probe was used to detect Mitochondrial membrane potential change by flow cytometry.NAC or?and?BAPTA-AM was used to pretreat cells to discuss the relationship between endoplasmic reticulum and mitochondrial apoptotic pathways in HO-1 siRNA-mediated apoptosis.Results 1.Low HO-1 expression in CML patients may indicate complete molecular remission.The expression of HO-1 was elevated with the progression of CML from chronic phase to accelerated phase and then blast phase or its relapse.CML underwent molecular remission and relapse,as suggested by the correlation between HO-1 expression and the count of primitive CML cells.2.pRNAi-siHO-1-GFP managed to effectively targeted-silence the expressions of HO-1.The survival rates of various BMMNC groups were related with DNR concentration time-and dose-dependently.The survival of the pRNAi-siHO-1-BMMNC group was evidently inhibited,with the apoptotic rate being significantly higher than those of the other two groups at each time point.Besides,compared with the other two groups,HO-1 mRNA and protein expressions in the pRNAi-siHO-1-BMMNC group were down-regulated,whereas those of caspase3,caspase8 and caspase9 were up-regulated.Kasumi-1 cells were successfully inoculated into nude mice.The Kasumi group succumbed to tumors faster than the pRNAi-siHO-1-Kasumi group did,and the tumors grew at a higher speed into larger volumes.Kaplan-Meier survival curves showed that the pRNAi-siHO-1-K group survived longer than the Kasumi group did after tumor formation.In the blood samples collected from the tail veins,the counts of leukocytes and platelets and hemoglobin levels in the Kasumi group were lower than those of the pRNAi-siHO-1-K group.The AML1/ETO was overexpressed in bone marrow,liver and spleen,which was higher in Kasumi group than that in pRNAi-siRNA-K group.Pathological test:HO-1 siRNA could relieve the infiltration of inflammatory cells.3.The expression of HO-1 was successfully silenced by pRNAi-siHO-1-GFP.The transfection efficiency observed after 72h was 95.78%.Real-time PCR and Western blot showed that the silence rate of HO-1 reached over 75%.DNR inhibited the proliferation of Kasumi-1 cells in a dose and time-dependent manner,and pRNAi-siHO-1-GFP remarkably inhibited the survival of Kasumi-1 cells.Down-regulation of HO-1 was conducive to cell apoptosis through caspases activation?cleaved-caspase3,caspase8 and caspase9?.The activation of caspase12,Ca²⁺accumulation as well as ROS generation,release of Cyto C and reduction of MTP indicated that endoplasmic reticulum apoptotic pathway and mitochondrial apoptotic pathway were involved in the cell apoptosis mediated by HO-1 siRNA.To block ROS generation or?and?Ca²⁺ accumulation by NAC and BAPTA-AM,the expressions of apoptosis-related genes decreased,ROS generation and Ca²⁺ accumulation were inhibited.Besides,the cell apoptosis was suppressed.Conclusion 1.HO-1 is a potential molecular indicator for the progression and relapse of CML.2.Lentivirus-mediated HO-1 gene silencing may promote the apoptosis of human M2 leukemic cells by activating caspase-dependent apoptotic pathways.Targeted silencing of HO-1 expression can inhibit the proliferation and infiltration of leukemic cells in nude mice and thus prolong their survival.The findings provide valuable experimental evidence for the molecular targeted therapy of M2 leukemia.3.The endoplasmic reticulum and mitochondrial apoptotic pathways were activated through HO-1 siRNA.At the same time,Ca²⁺ was more prone to accumulation and ROS was more easily generated,while mitochondrial transmembrane potential was reduced.Thus,Cyto C was released from mitochondria to cytoplasm and caspases were activated for the following cascade to facilitate the apoptosis of Kasumi-1 cells,during which Ca²⁺ and RIS interacted and were interdependent,thus affecting the activation of apoptotic pathways.