| Background and ObjectivesAcute myeloid leukemia (AML) is a malignant proliferative disease that occurs in hematopoietic stem/progenitor cells. AML is characterized by unlimited proliferation of leukemia cells originating from the myeloid lineage, and often accompanied by infiltration in extramedullary tissues. The mortality rate of leukaemia is very high. With the development of chemotherapy regimens, the induction remission rate of leukemia has been significantly improved, but the recurrence rate is still high. Exploring the reasons, the extramedullary infiltration of leukemia cells is one of the important cause.Thirty to forty percent of newly diagnosed AML patients present with extramedullary infiltration (EMI), of which common sites include lymph nodes, skin, liver, spleen, gingiva, and central nervous system. EMI is reported to be more common in M4/M5 subtypes (FAB classification) and associated with poorer treatment response,especially in AML patients with the t(8;21) translocationEZH2 known as the catalytic subunit of polycomb repressive complex 2 (PRC2), has intrinsic histone methyltransferase activity. EZH2 can repress its target genes (such as tumor suppressor gene and cyclin protein) transcriptionally by trimethylation of lysine 27 on histone H3 (H3K27). thereby promoting proliferation and aggressiveness of cancer cells. Overexpression of EZH2 has been found in a variety of human malignancies, including prostate cancer, breast cancer, lung cancer, nasopharyngeal cancer, liver cancer, kidney cancer, colorectal cancer and is closely correlated with tumor aggressiveness, metastasis and poor prognosis. Current research shows that EZH2 overexpression in high-risk myelodysplastic syndrome (MDS) and AML transformed from MDS and confers poor prognosis. In addition, high EZH2 has been found to be associated with high lactate dehydrogenase (LDH), high WBC count and poor prognosis in bone marrow tissues from patients with de novo AML.Our study group has found that the mRNA and protein expression levels of EZH2 in AML patients were both significantly higher than in ITP patients. Furthermore, a positive correlation between EZH2 mRNA expression and percentage of peripheral blood blasts was found in AML patients (r=0.404, p=0.009). The migratory capacities of Kasumi-1 and HL-60, which both show a high level of EZH2 expression, were markedly higher than those of U937 and KG-1α.Through the above research,we speculated that EZH2 may be associated with leukemia cells EMI in AML. However, whether and how EZH2 is involved in the EMI process of leukemia cells remains largely unexplored.Therefore, to this end, in order to further explore the relationship between EZH2 and EMI in AML.we used EZH2 knock-down to investigate this gene’s role in the growth apoptosis and migration of Kasumi-1 cells, and the associated molecular mechanisms. To provide theoretical basis for the prevention and treatment of leukemic marrow infiltration.Contents and methods1 Construction of plasmid vectors, packaging vires and transfecting target cellBased on the principles of designing siRNA, we design three shRNA sequences (siRNA-1,siRNA-2 and siRNA-3) to knockout EZH2 expression against the common area of EZH2 gene transcription. Construct lentiviral vectors to carry shRNAs to knockdown the expression of EZH2 perpetually in kasumi-1 cells. We used flow cytometry to detect transfection effect and we selected the GFP-positive cells for culturing with flow cytometry. At last, QRT-PCR and WB were used to measure mRNA and protein expression levels of EZH2, respectively, We choose the highest efficiency of EZH2 silencing to perform subsequent experiments (henceforth termed the siEZH2 group).2 The effect of biological characteristics by knockout EZH2 on kasumi-1 cells.In our study, the wild kasumi-1 defined as wild group, transfection with scramble sequence of kasumi-1 was defined as NC group, and transfection with interference EZH2 gene sequences of cells are defined as siEZH2 group. Then QRT-PCR and WB was performed to virify the effect of EZH2 knock out. To investigate whether a high level of EZH2 is associated with disease aggressiveness in AML, we used the CCK.8 assay and flow cytometry to measure proliferation and apoptosis of kasumi-1 cells respectively. And we performed a cell migration assay to investigate the migration ability of three groups of cells.3 Explore the mechanism of EZH2 involved in cell migrationTo investigate the possible mechanism via which EZH2 is involved in migration, Based on the metastasis of EZH2 in solid cancer. we measured the level of several proteins (such as ERK、p-ERK、c-myc、p-cmyc、MMP-2 and E-cadherin) that may associated with these processes by WB.4 Statistical analysisStatistical analysis was performed with the statistical package SPSS 19.0. Data from this study were reported as mean±standard deviation, and one-way ANOVA was used to assess the values and percentages between groups. P values<0.05 were considered significant. Levene test was used to detect the homogenety of variance, if the variance is homogeneity, LSD method was used for multiple comparison; if not, using Welch instead of One-Way ANOVA to analysis of the variance, and then using Dunnett’S T3 for multiple comparisons among the groups.Results1 Construction plasmid vector, packaging virus and transfection kasumi-1 cells.Based on the principles of designing siRNA, we designed three shRNA sequences (siRNA-1, siRNA-2 and siRNA-3) to knockout EZH2 expression against the common area of EZH2 gene transcription. Synthesised these sequence to Oligo, and then connected to carrier. After that, transformed vectors into E.eoli, then plasmid was extracted, at last, gene sequencing was sequenced. By the sequencing results, we found the three pGFP-shEZH2 sequences inrecombinant plasmids were matched with the design shRNA sequence and correctly inserted into the the pGFP viral vectors. They could be used in the next experiments. We used the three packaged plasmid to transfect 293T cells. We observed cells with fluorescence under a microscope after transfected 48h. We found the visible fluorescence decreases when increased the dilution factor. And we took photos of the cells with fluorescence microscope.Then we used flow cytometrye to detect the transfection rate of the four groups. The transfection rate of siRNA-1 group, siRNA-2 group, siRNA-3 group and NC group were 77.12%,48.78%,71.72%and 76.41%, respectively. We selected the GFP positive cells for culturing with flow cytometrye.The gene silencing effects were confirmed by QRT-PCR and Western Blot. We chose the most obvious group of down-regulated EZH2 as siEZH2 group. We found that EZH2 mRNA expression was significantly down-regulated following exposure to siRNA-1 (0.0096±0.0004, P=0.000), siRNA-2 (0.0285±0.0031, P<0.001) and siRNA-3 (0.0070±0.0002; P<0.05). EZH2 protein expression level was also down-regulated in these three groups. We found that siRNA-3 produced the highest efficiency of EZH2 silencing, therefore, we choose siRNA-3 with which to perform subsequent experiments (henceforth termed the siEZH2 group), mRNA expression levels of EZH2 were significantly reduced in siEZH2 group compared to wild and NC group (0.1243±0.0074,0.1147±0.0091and 0.0071±0.0002 for the expression of EZH2 mRNA in wild、NC and siEZH2 groups, respectively; p<0.001).2 The biology behavior of kasumi-1 after knockdown of EZH22.1 The effect on proliferation after knockdown of EZH2To investigate whether a high level of EZH2 is associated with disease aggressiveness in AML, we used the CCK8 assay to measure proliferation in three group cells. We observed the time of five different time (0h,24h,48h,72h and 96h),then we use time as the abscissa, cell survival as the ordinate to draw a picture, we found that proliferative capacity was significantly lower after knocking down EZH2.2.2 The effect on apoptosis after knockdown of EZH2In this study, The Annexin V-PI Apoptosis Detection kit was used to detect and quantify apoptosis by flow cytometry. The result showed that the ratio of early apoptosis in siEZH2s group[(6.23±0.38)%] was higher than in wild group [(3.14±0.37)%, P=0.001]and NC group [(2.90±0.33)%, P=0.001]; and the ratio of later apoptosis in siEZH2 group [(20.77±1.15)%] was higher than in wild group [(3.77±0.36)%, P=0.001]and NC group [(4.70±0.85)%, P=0.000]. The result showed the apoptosis is increased after knockdown EZH2.2.3 The effect on capacity of cell migration after knockdown of EZH2We performed a cell migration assay to investigate the relationship between migration and EZH2 in AML cells. In our study, a transwell migration chamber was used to test the migration ability of three group cells. we found that the cell number in lower chamber of siEZH2 group[(3.50±0.25)%] was significantly reduced compared to the wild group[(9.08±0.38)%, P=0.000] and NC group[(9.17±0.38)%, P=0.000]. When the ratio of migrated to non-migrated cells was compared, the cells in the siEZH2 group showed a significantly lower ratio than either of the control group (p<0.05). Finally, the siEZH2 group (26.67±1.53) had markedly fewer cells on the membrane than wild group(73.33±3.51, P=0.000) and NC group(72.00±3.00, P=0.000). Therefore, our data suggest that EZH2 may be involved in the migration of AML cells, thus supporting a role for EZH2 in EMI in AML patients.3 The possible mechanism via which EZH2 involved in migrationTo investigate the possible mechanism via which EZH2 is involved in migration, we measured the level of several proteins associated with these processes by WB. We found that H3K27me3 is remarkably down-regulated by knockdown of EZH2. In addition, p-ERK、p-cmyc and MMP-2 are significantly reduced, while levels of ERK、c-myc and APP remain essentially unchanged. Furthermore, E-cadhering, a protein associated with invasion and metastasis of malignant tumors is obviously up-regulated. These findings suggest that EZH2 may modulate migration via the signaling pathway of p-ERK/p-cmyc/MMP-2 and E-cadherin in AML cells.Conclusion1 SiRNA can down-regulated the expression of EZH2, we can get a permanent down-regulated of EZH2 by the technique of RNAi and lentivirus transfection。2 Knockdown of EZH2 induces apoptosis and reduces proliferation and migration ability of AML cells3 EZH2 may modulate migration via the p-ERK/p-cmyc/MMP-2 signaling pathway and E-cadherin in AML cells. |
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