Mechanism And Functional Analysis Of CRM1 And MYBL2 Genes In Acute Myeloid Leukemia

Part one.Effects and Mechanism of CRM1 genne in Acute Myeloid LeukemiaObjective:To study the role of chromosome region maintenance 1?CRM1?in acute myeloid leukemia and investigate the apoptosis effect and molecular mechanism of its inhibitor KPT-330 on human acute myeloid leukemia cells.Methods:The expression of CRM1 in leukemia cell lines and non-leukemia bone marrow cells was detected via Western blot.We used KPT-330 with different concentrations incubate cells to inhibit the expression of CRM1 and Cell Counting Kit-8assay to quantify cells proliferation differences after exposure to KPT-330 for 24 hours.Fluorescence microscope was used to observe the morphology of leukemia cells when treated with KPT330 24 hours.Annexin V-FITC/propidium iodide staining followed by flow cytometry was used to detect the apoptosis and the cell cycle of leukemia cells.Then,Western blot was used to assessed the following well-recongnized markers of apoptosis:Caspase-9,Caspase-3 and PARP.Long noncoding RNA?LncRNA?microarray was used to analyze the differentially expressed genes from the cluster analysis.Results:Our study showed that CRM1 had a high level expression in variety of leukemia cells including NB4,K562,HL-60,CCRF,but not in normal bone marrow.KPT-330 could significantly decrease the expression of CRM1 and inhibite proliferation of leukemia cells in a dose-dependent manner.Compared with the control group,abnormal cells were observed under fluorescence microscopy when CRM1 was decreased.In addition,inhibiting CRM1 induced apoptosis.The apoptosis rates of NB4?K562 and HL-60 were respectively?4.95±0.21?%,?14.05±0.07?%,?4.95±0.63?%?0.2?mol/L 24h?;?6.10±0.56?%,?20.75±0.21?%,?5.85±0.07?%?0.2?mol/L 48h?;?41.15±0.21?%,?35.45±0.35?%,?15.65±0.07?%?1.0?mol/L 24h?;?52.45±0.35?%,?47.45±2.47?%,?16.80±1.98?%?1.0?mol/L 48h?,and the differences were statistically significant compared with control group?P<0.05?.Cell cycle assay showed that G₂ phase cells were obviously reduced and subG₁ phase cells were increased after CRM1 was inhibited by KPT-330.Moreover,the expression levels of cleaved caspase-3,cleaved Caspase-9 and cleaved PARP were increased.LncRNA microarray analysis showed that many mRNAs and lncRNAs differentially expressed compared with control.The involved pathways in which these differentially expressed genes included Glutathione metabolism and Toll-like receptor signaling pathway.Conclusions:CRM1 plays an important role in the proliferation of acute myeloid leukemia cells,and inhibiting CRM1 can lead to leukemia cell apoptosis.KPT-330 can effectively inhibit proliferation and promote apoptosis of leukemia cells,which induced apoptosis is partially related to Glutathione metabolism and Toll-like receptor signaling pathway.Part two.Preliminary study on the mechanism of MYBL2 gene in acute myeloid leukemia cellsObjective: To investigate the expression of MYBL2 gene in the human acute myeloid leukemia cell lines and explore its effects and mechanisms of cell proliferation and cell cycle after silenced.Methods: Western blot was used to detected the protein expression of MYBL2 in several leukemia cell lines.Si RNA lentivirus was used to interfere expression of MYBL2 in NB4 and K562 cells,which had high expression of MYBL2.Si-MYBL2 group: the expression of MYBL2 in NB4 and K562 cells was inhibited by MYBL2-Si RNA lentivirus.Si-NC group: NB4 and K562 cells were infected by negative control Si RNA lentivirus.Fluorescence microscope was used to observe the morphology of leukemia cells between interference group and the negative control group.Proliferation differences were detected by CCK8 test.Flow cytometry was performed to assessed the cell cycle of leukemia cells after propidium iodide staining in two groups.The nucleus by hoechst 33342 staining was observed in leukemia cells.We identified the expression of MYBL2 protein after Si RNA lentivirus infection via western blot.Furthermore,the recognized cell cycle markers that C-MYC and PLK1,and the phosphorylation level of ERK were measured by western blot in the Si-MYBL2 and Si-NC groups.Results: MYBL2 protein was highly expressed in NB4,K562 and other leukemia cells.When MYBL2 gene of leukemia cells was successfully interferred,the cell volume of the experimental group was significantly increased compared with that of the control group.CCK8 assay showed that cell proliferation decreased obviously after MYBL2-Si RNA lentivirus infected leukemia cells 96 hours?P<0.05?,the difference was statistically significant compared with Si-NC group.Downregulating MYBL2 induced cell cycles arrest in G1 phase?P<0.05?and prevented cell cycle progression,the difference was statistically significant.Hoechst 33342 staining showed that nucleus became larger and irregular in the Si-MYBL2 group.These remindered that the MYBL2 might promote leukemia cells proliferate by regulating cell cycle.Western blot show that the expression of C-MYC and PLK1 in leukemia cells was inhibited,which was in accordance with MYBL2 protein expression,while the phosphorylation of ERK was significantly increased.Further studies should be conducted to explore the possible mechanism of MYBL2 in leukemia cells.Conclusion: In vitro,MYBL2 gene plays an important role in the proliferation of leukemic cells and affects the cell cycle distribution;Inhibiting MYBL2 gene induces cell cycle arrest in G1 phase and cell proliferation significantly decreased.MYBL2 gene can regulate cell cycle in leukemia cells with C-MYC and PLK1 genes.The MYBL2 gene may regulate cell cycle of leukemia cell through ERK cell signaling pathway.