Study Of Cell Proliferation In Acute T Cell Leukemia With High Expression Of Notch1 Gene

Background:Acute T lymphoblastic leukemia(T-ALL)is an aggressive malignancy with the accumulation of immature T lymphoblastic progenitor cells.It accounts for 20% of all cases of acute lymphoblastic leukemia(ALL),especially occurred in children and adults over 40 years old.The clinical characteristics of T-ALL patients include high white blood cells,easy to infiltrate the nervous system and the presence of a mediastinal mass.Although the treatment of T-ALL has made some progress,there are still about 20% newly diagnosed children and 50% adults faliure to induction chemotherapy or faced with recurrence,even if the application of high intensity chemotherapy.Only less than 50% of the patients can obtain longer survival.Therefore exploiting new targeted therapies has been the focus of research in recent years.According to the statistics,about 70% of patients carried mutations in the Notch1 gene.The Notch1 receptor encoded by mutation of the Notch1 gene can combine a variety of ligands,and then regulate the expression of downstream genes,such as Myc oncogene.Not only that,Notch1 gene participated in the proliferation and differentiation of tumor cells,T-ALL cells and other metabolic activities.So the Notch1 mutation and its mechanism in facilitating proliferation are always the research focus of T-ALL.In our previous study,in order to explore the role of Notch1 in T-ALL,Notch1 transgenic zebrafish has successfully established,and through gene chip detection,screening of differentially expressed genes found that transgenic fish significantly higher expression of CDK4 and CDK6 genes,compared with normal zebrafish.Does CDK4/6 play a role in the pathogenesis of T-ALL with high expression of Notch1.Can the CDK4/6 selective inhibitor Palbociclib,which approved by FDA,reverse the partial proliferation.Objective:We hypothesized that the Notch1 signaling pathway and cell cycle related genes are closely related.so the application of zebrafish in vivo and acute T cell lymphoblastic leukemia(T-ALL)cells for in vitro study help us understand related mechanism of the proliferation of leukemia cells with the high expression of Notch1.And the cell cycle inhibitor was applied in both study.This thesis is mainly divided into two parts:(a)in vivo research of T-ALL,the transcriptome sequencing of Notch1 transgenic zebrafish suggest high expression of cell cycle related genes and signal pathway of Notch1 gene;(b)in vitro research,the selective cell cycle inhibitor,Palbociclib,lead leukemia cells G1 phase arrest and occur proliferation obstruction.Methods:For the research of the specific mechanism of Notch1 signaling pathway in T-ALL,in vivo study,Notch1 transgenic zebrafish and wild type zebrafish were selected as the experimental group and control group.The differential gene expression analyzed by transcriptome sequencing technology suggested highly expression of cell cycle genes.And targeted inhibitor on differential expression genes was applied in vivo study,RT-qPCR method to detect the expression of related cytokines.In vitro study,lymphocytic leukemia cell lines(A3,MOLT-4)with high expression of Notch1 and normal human peripheral blood mononuclear cells(Peripheral blood mononuclear cell,PBMC)was the experimental group and control group respectively.First conventional PCR method was applied to detect the expression of Notch1 signal and cell cycle related genes in two groups.CCK8 method was used to detect the inhibition rate of Palbociclib on PBMC cells and T-ALL.Then the cell cycle and proliferation were analyzed by multiple parameter flow cytometry of two groups of cells in different concentrations of Palbociclib(0,50,100nM).And morphology of cells were observed by cell smear.RT-qPCR detection method can detect expression of related Cytokines in different concentration of Palbociclib under the control of Notch1 genes.Results:In Notch1 transgenic zebrafish,the chip indicated the high expression of cyclin kinase CDK4/6.RT-qPCR detected the decreased expression of CDK4/6 treated by Palbociclib on zebrafish.Conventional PCR detection also highly expressed CDK4,CDK6,Myc,Notch1 in T-ALL cell lines.The IC50 values of Palbociclib on A3 and MOLT-4 cell lines were(0.43 ± 0.12)nM and(0.10± 0.16)M respectively.While the IC50 of Palbociclib on PBMC is(0.83 ± 0.15)nM,significantly higher than the T-ALL cell line(P < 0.01).Cell cycle analysis by flow cytometry suggested that the number of(S+G2)phase of A3,MLOT-4 were(55.93 + 1.18)% and(58.17 + 2.40)% respectively,higher than that of the control group,which is(48.93 + 1.85)%).After treatment of Palbociclib,the number of(S+G2)phase of two groups of were all decreased in a concentration dependent manner(P < 0.05).In 50 nM,the cell number for multiplication of both group had no difference(P > 0.05).Quantitative detection of gene expression by RT-qPCR indicated that the expression of CDK4/6 in T-ALL cells after treatment by Palbociclib(50,100nM)was decreased,compared with the concentration of 0nM.In he concentration of 50 nM,the transcription of Myc,Notch1 has no significant difference with 0nM.The expression of Myc gene was decreased,and that of Notch1 gene was interestingly increased in 100nM(P < 0.05).Coclusions:T-ALL cells highly expressed CDK4/6,Myc gene,common in Notch1 transgenic zebrafish.Palbociclib can not only lead tumor cells arrest in G1 phase and proliferation inhibition inhibiting CDK4/6,and also can inhibit the expression of downstream Myc gene to inhibit tumor cell proliferation,indirectly against the promotion of tumor cells caused by Notch1 activation.