| Background and ObjectivesLeukemia is a malignant tumor which is involving hematopoietic stem and/or progenitor cells. The incidence of leukemia in China is417per100000person, it is located number seven in the tumor list because of its high rate of mortality. With the improvement of chemotherapy, leukemia’s remission rate of induction has been significantly improved, but the relapse rate is still high as30%-40%. Digging the causes, extramedullary infiltration of leukemia cells is the important reason that leads to relapse and death.Acute myeloid leukemia with t (8;21)(q22; q22) to form AML1/ETO fusion gene, is a type of AML accounting to12%. Foreign scholars believe that AML1/ETO+is a good factor of prognosis, while Chinese scholars do not agree with that. They find that acute leukemia patients with t (8;21) abnormal and accompanied with extramedullary infiltration come to poor prognosis. Extramedullary leukemia, especially Central Nervous System Leukemia (CNSL) has bad effect on efficacy and prognosis, especially in AML1/ETO+leukemia patients. CNSL is an obstacle in treating patients, leading to leukemia relapse. There is no way to monitor extramedullary leukemia at present. Therefore, to understand the mechanism of extramedullary infiltration in leukemia is the issue should be solved, and it is related with searching for new therapeutic targets.APP gene is located on21q21.3, about300kb, containing19exons. APP protein is a type I transmembrane protein which is widely expressed in various types of cells, relating with cell growth, apoptosis, and it is also involved in gene transcription, calcium balance. APP gene is overexpressed in pancreatic cancer, oral squamous cell carcinoma, colorectal cancer and other solid tumors, and it can promote the proliferation of cells in some malignant tumors such as pancreatic cancer, oral squamous cell carcinoma, thyroid cancer and colorectal cancer. Overexpression of the APP gene is a poor prognostic factor in oral squamous cell carcinoma. However, the biological function and role of the APP gene in tumor development is not clear. Also, the results of the APP gene in the blood tumor are come from Gene expression screening, and we didn’t do further research on leukemia.Our clinical data indicated that in acute leukemia patients with AML1/ETO+, the incidence of extramedullary leukemia especially in central nervous system in APP overexpressed patients was significantly higher than those with low expression, along with short overall survival time and recurrence-free time, these datas were statistically significant (p<0.05). Wang Wei using QRT-PCR to detect APP expression in acute myeloid cell lines such as kasumi-1, HL-60, HL-60/ADM and U937, and the result showed APP mRNA was the highest expression. Based on these findings, we use Kasumi-1cells as our subject and virus vector as our tool to interfere APP expression in kasumi-1cells, and then to study its biological function in leukemia cells. Clarify the mechanism of APP gene involved in extramedullary leukemia to provide a theoretical basis for a new therapies on precaution extramedullary leukemia.Contents and methods1. Kasumi-1cells are the model of AML1/ETO+leukemiaInorder to determine kasumi-1cell is a cell line of acute myeloid leukemia, we use some methods to detect it’s marks. First, we use wright’s stain and peroxidase stain to detect it’s morphological characteristics. Second, Flow cytometry was used to detect molecular markers on kasumi-1cells’ surface. Thirdly, FISH and karyotype analysis to verify kasumi-1cell is with t (8;21) abnormal and AML1/ETO+fusion gene.2. Construction of plasmid vectors, packaging virus and transfecting target cellAccording to the principles of designing siRNA, we design three shRNA sequences to interfer APP expression against the common area of APP gene transcripts, such as siRNA-1, siRNA-2and siRNA-3. Construct Lentiviral vectors to interfere APP expression permanently in kasumi-1cells which are overexpressing of APP. We selecte GFP-positive cells by Flow cytometry, and then cultured in vitro. At last, we use fluorescence quantitative PCR and Western-Blot to detect the effect of APP interference, and select the best effect of interference of the three and cultured these cells as target cells in vitro for the next Follow-up experiments.3. The research about function of APP gene in leukemia cells(1) Groups:wild group (wild), transfected with a scramble sequence as negative control group (NC), transfected the shRNA to interfere APP expression as target group (siAPP).(2) Determin the effect of interferenceUse Fluorescence Quantitative PCR and Western-Blot technology to detect the expression of APP in these three groups of cells.(3) The effect of biological characteristics after inhibiton of APP expression on leukemia cellsTo explore the biological function of the APP gene in leukemia cells, we use CCK-8assay, flow cytometry and Transwell experiments to observe kasumi-1cells changes on cell proliferation, apoptosis and migration after inhibition of APP expression.4. Preliminary research on APP-mediated mechanismBased on our clinical data which shows that APP gene is closely related with extramedullary leukemia, we use the fluorescence quantitative PCR and Western-Blot to detect molecules which are related with migration, such as CXCR4, MMP-2, ERK, MMP-9, and c-Myc. At last, we are looking for an molecular signal transduction of APP-mediated as the mechanism on leukemia extramedullary infiltration.5. Statistical analysisWe use SPSS13.0statitical sofrware to analysis data. One-Way ANOVA is adopted to compare the values among the groups, description of data as x±s, p<0.05considered statistically significant. Levene test detect the homogenety of variance, if the variance is homogenety, then we use LSD method for multiple comparison; if not, using Welch instead of One-Way ANOVA to analysis of the variance, and then using Dunnett’s T3for multiple comparisons among the groups.Results1. The genetic characteristics of Kasumi-1cells(1) PCR and WB detect the expression level of APP on Kasumi-1cellsOne-way ANOVA analyze the APP mRNA expressions in kasumi-1, HL60and U937AML cell lines are0.1319±0.0068,0.0380±0.0049and0.1810±0.0077, APP mRNA is the highest in kasumi-1. Levene test detect the homogenety of variance, F=0.481, p=0.640; One-Way ANOVA to analysis of the variance and showes F=300.989, P=0.000. Then we use LSD method for multiple comparison among the groups, p=0.000between each group. The result of Western-Blot is consistent with PCR. The band of APP protein in kasumi-1cells is darker than the other two, and the band in U937cells is the lightest.(2) The cell line of kasumi-1has morphological characteristics of myeloid leukemiaAfter Wright’s staining, we use microscope to detect the morphological charactersitics of kasumi-1cells. It can be observed a large number of immature cells which has big nucleus and little cytoplasm, and darker chromatin granules are visible. We also can see brown substance deposited in the cytoplasm under peroxide enzyme staining. Therefore, these morphological characteristics suggest kasumi-1cells are the cells from myeloid leukemia.(3) Cytogenetic features of kasumi-1 Fluorescence in situ hybridization (FISH) assay shows kasumi-1cells have strong signals of AML1/ETO fusion gene, and the ratio of cells with AML1/ETO+is about66%.Karyotype analysis shows kasumi-1cell is79,XX,t(8;21), besides, it also has number abnormal and2p-、7q-、5q-、13q+、1-3mar.(4) Flow cytometry detect cell markersResult shows kausmi-1cell molecular markers as follow, CD33:74.94%, CD38:1.26%, CD34:31.74%, CD117:59.89%, CD13:97.11%, CD4:1.13%, CD71:38.94%, CD15:11.38%. So, we conclude kasumi-1cell is CD33+/CD38-/CD34+/CD71+/CD117+/CD4-/CD13+/CD15-,according to morphological characteristics and cytogenetic features, we can tell kasumi-1cell has the signature of the myeloid lineage.2. Package viruses and transfecting target cells(1) Design the sequence to interfere APP and sequencing the recombinant plasmidAccording to the principles of siRNA designing, we design three shRNA sequences to interfer APP expression against the common area of APP gene transcripts. Synthesis these sequence to Oligo, and then connected to carrier. After that, vectors were transformed into E. coli, then, plasmid was extracted and sequenced. Sequencing results shows that:these three pGFP-shAPP sequences in recombinant plasmids are matched with the design shRNA sequence and correctly inserted into the the pGFP viral vectors. They can be used in the next experiments.(2) virus harvestUsing liposome to transfect the three packaged plasmid into293T cells.48h later, we can observe cells with fluorescence under a microscope and the visible fluorescence decreases with increasing dilution factor.(3) viruses infect target cellsInorder to get the optimal infection conditions for Kasumi-1cells, we perform pre-experiment according to the Manuscript of the lentivirus virus transfection. Flow cytometry was used to detect the transfection rate after72h when lentivirus transfected kasumi-1cells. In the NC group, the transfection rate was91.4%, the siRNA-1group was66.8%, the siRNA-2group was83.2%, and the transfection of siRNA-3group was86%. At the same time, we filter1×105GFP-positive cells for each group, and cultured in vitro for subsequent experiments.(4) Select the most effective interference sequenceFlow cytometry filters GFP+cells each group and culture in vitro. After72h, we can observe kasumi-1cells grow in groups, and these cell are green fluorescence. Quantitative PCR detects APP expression in group siRNA-1, siRNA-2, siRNA-3as0.0144±0.0003,0.0047±0.0007,0.5778±0.0009respectively. Levene test the variance is homogenety, F=2.860, p=0.134; We use LSD method for multiple comparison, the results are p=0.000between groups, and the APP mRNA is the least in group siRNA-2. The result of Western-Blot is consistent with PCR. The band of APP protein in group siRNA-2is lightest than the other two. Therefore, we selecte kasumi-1cells which are transfected with siRNA-2sequence as the experimental interference group (siAPP group).We selecte wild kasumi-1cells as blank control (wild) and transfected scrmble sequence as a negative control (NC), using PCR and western-Blot to detect the interference effect of siRNA on APP expression. PCR results show that relative expression of APP mRNA expression in wild, NC and siAPP group are0.1265±0.0053.0.1230±0.0030and0.0049±0.0006. Levene test the variance is homogenety, One-way ANOVA analysis of the variance F=1146.989, p=0.000; We use LSD method for multiple comparison, p=0.000when group siRNA compare with group NC or group wild, while there is no statistically significant difference between group wild and group NC (p=0.271). Western-Blot results show APP protein band in siAPP group is significantly weakened than wild and NC groups, while there is no significant difference in NC group and wild group. That is to say, we have successfully interfere APP expression on Kasumi-1cells.3. Biological function of APP gene in leukemia cells(1) The effect on apoptosis after interferance of APPFlow cytometry is used to detect the proportion of early apoptosis and late apoptotic cells in each group. The results show as follows:the proportion of early apoptotic in wild group, NC group and siAPP group are21.43±0.86,21.67±0.78, and29.00±0.98respectively. Levene test the variance is homogenety, One-way ANOVA analysis of the variance F=71.927, p=0.000; Using LSD method for multiple comparison, group siRNA compare with group NC and group wild, p=0.000respectively. When group wild compare with group NC, there is no significant difference (p=0.756). That means early apoptosis is significantly increase after interferenc of APP. The proportion of late apoptosis in wild group, NC group and siAPP group are12.33±0.75,12.90±1.25,19.80±1.51respectively. Levene test the variance is homogenety, One-way ANOVA analysis of the variance F=35.239, p=0.000; Using LSD method for multiple comparison, group siRNA compare with group NC and group wild, p=0.000respectively. When group wild compare with group NC, there is no significant difference (p=0.588). That means late apoptosis is significantly increase after interferenc of APP.(2) The effect of APP in kasumi-1cells on cell cycleWe use Flow Cytometry to detect the cell cycle distribution on wild group, NC group and siAPP group. The ratio of G0/G1, S and G2/M in group wild are36.97±9.12、26.03±5.20,34.93±2.80respectively; and36.40±8.17,26.27±4.8634.80±7.56in group NC; and36.63±3.90.28.37±6.27,33.17±2.42in group siRNA.Levene test the variance of G0/G1is homogenety, F=1.252, P=0.351, One-way ANOVA analysis of the variance F=0.004, p=0.996; Using LSD method for multiple comparison, p=0.958when group siRNA compare with group wild, p=0.971when group siAPP compare with group NC, and p=0.929when group wild compare with group NC. That means there is no significant difference on cell cycle of GO/G1.Levene test the variance of S is homogenety, F=0.063, P=0.939, One-way ANOVA analysis of the variance F=0.165, p=0.851; Using LSD method for multiple comparison, p=0.620when group siRNA compare with group wild, p=0.655when group siAPP compare with group NC, and p=0.960when group wild compare with group NC. That means there is no significant difference on cell cycle of S. Levene test the variance of G2/M is homogenety, F=4.654, P=0.060, One-way ANOVA analysis of the variance F=0.123, p=0.887; Using LSD method for multiple comparison, p=0.672when group siRNA compare with group wild, p=0.695when group siAPP compare with group NC, and p=0.974when group wild compare with group NC. That means there is no significant difference on cell cycle of G2/M.(3) The effect of cell migration after the interference APP expressionWe use transwell chamber to detect the migration changes after APP gene silence. We can see cell density in the lower chamber of group siRNA is significantly reduced under microscope, and we caculate the migration rate of each group. Levene test the variance is homogenety, F=1.642, P=0.270, One-way ANOVA analysis of the variance F=16.107, p=0.004; Using LSD method for multiple comparison, group siAPP compare with group NC and group wild, P value is0.002and0.003respectively, while p=0.641when group wild compare with group NC.The cell number is significantly reduced in group siAPP under the microscope after staining the membrane of Transwell products. We choose5fields randomly to count cell number, and Levene test the variance is homogenety, F=0.379, P=0.692, One-way ANOVA analysis of the variance F=66.650, p=0.000; Using LSD method for multiple comparison, siAPP compare with group NC and group wild, p value are both0.000, while p=0.936when group wild compare with group NC.These datas reveal that cell migration ability is significantly reduced after APP silence.(4) The effect of cell proliferation after APP silenceWe use CCK-8assay to detect changes in cell proliferation after interference of APP. We observed24h,48h,72h and96h, and the results show that there is no significance in OD values of the cells in each group (p>0.05). The growth curves of the three groups come to together, and we can not see a difference. It means that interference of APP expression has no effect of proliferation on Kasumi-1cell.(5) Structure changes of Kasumi-1cell after APP silence We use scanning electron microscopy to observe the membrane structure of kasumi-1cell. There is rich of microvilli stout and the free end of the microvilli is enlargement to form a blunt rounded contours in NC group cells. The number of microvilli is significant reduced and become slender, pointed in siAPP group.4. The preliminary research of APP-mediated mechanism(1) PCR and WB detect MMP-2, MMP-9and CXCR4expressionWe get the Ct value from each sample of MMP-2, MMP-9and CXCR4according to amplification curve. β-actin as an internal comparison,△Ct value of each sample is calculated and using the2-△Ct as reletive mRNA expression of MMP-2, MMP-9and CXCR4. The relative expression level of cells in each group are calculated. The results show that MMP-2mRNA in group wild, NC and siAPP are0.1570±0.0317,0.1736±0.0462,0.0062±0.0013respectively. Levene test the variance is without homogenety, F=7.283, P=0.025, so we choose Welch to detect the variance, F=73.359, p=0.005; Using Dunnett’s T3method for multiple comparison, p=0.001when group siAPP compare with group NC, p=0.000when group siAPP compare with group wild, and p=0.996when group wild compare with goup NCMMP-9mRNA in group wild, NC and siAPP are (2.8800±0.2883)×10-7,(2.6833±0.7862)×10-7,(4.9433±1.9388)×10-7respectively. Levene test the variance is homogenety, F=2.691, P=0.146, One-way ANOVA analysis of the variance F=3.163, p=0.115; Using LSD method for multiple comparison, p=0.064when group siAPP compare with group NC, p=0.084when group siAPP compare with group wild, and p=0.850when group wild compare with goup NC.CXCR4mRNA in group wild, NC and siAPP are0.2391±0.0195,0.2280±0.0693,0.1744±0.0567respectively. Levene test the variance is homogenety, F=3.375, P=0.104, One-way ANOVA analysis of the variance F=1.283, p=0.344; Using LSD method for multiple comparison, p=0.185when group siAPP compare with group wild, p=0.261when group siAPP compare with group NC, and p=0.807when group wild compare with goup NC. While APP mRNA in group wild, NC and siAPP are0.1175±0.0406,0.1035±0.0283,0.0075±0.0012respectively. Levene test the variance is homogenety, F=3.461,P=0.100, One-way ANOVA analysis of the variance F=13.164, p=0.006; Using LSD method for multiple comparison, group siAPP compare with group wild and group NC, p value are0.003and0.006respectively, while p=0.571when group wild compare with group NC.We perform30ug protein samples for SDS-PAGE electrophoresis and β-actin as an internal comparison, MMP-2protein bands is seen in wild group and NC group while it is significantly reduced in siAPP group. But, CXCR4protein expression does not change significantly.(2) WB detecte MAPK signal transduction pathwayWe perform50ug protein samples for SDS-PAGE electrophoresis, and GAPDH as internal comparision. The results show that p-ERK and c-Myc protein expression is significantly reduce after interference of APP expression, while ERK protein does not change. Therefore, we speculate that APP through p-ERK/c-Myc/MMP-2signaling pathways mediated the leukemia cells in extramedullary infiltration.Conclusion1. Kasumi-1cells have genetic characteristics of AML1/ETO+, and this cell line belongs to myeloid leukemia cells.2. RNAi successfully interfere APP expression on kasumi-1cells, and we get a stably transfected cell lines.3. Interfere of APP expression, migration of kasumi-1cell is significantly reduced and cell apoptosis is significantly increased, while cell cycle and cell proliferation has no change.4. APP via MAPK signaling pathway such as p-ERK/c-Myc pathway to regulate the expression of MMP-2, which mediates leukemia cells in the development of extramedullary infiltration. |
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