Molecular Mechanisms Of Acute Myeloid Leukemia Induced By EVI-1 Gene

Objectives:Acute myeloid leukemia(AML)is a malignant clonal disease of hematopoietic stem cells that can occur with high heterogeneity and high mortality at all ages.The development of AML and the specific genes are associated with abnormal chromosome expression.The EVI-1 gene is located in the long arm 2 region 6 band 2subband of human chromosome 3(3q26.2),and the encoded EVI-1 protein is a nuclear transcription factor protein.EVI-1 protein is an important regulator of human hematological tumors and some solid tumors.It is closely related to the development of various acute leukemias and is also involved in mammalian embryo development.Multiple studies have shown the presence of EVI-1 gene overexpression in approximately 8% of AML patients.In AML,MM,and CML,EVI-1 gene overexpression has been associated as a poor prognostic factor and associated with disease-free survival in patients.At present,the molecular mechanism of EVI-1 in the development of AML in vivo and in vitro is not perfect,and there are few reports on the drug that can effectively inhibit EVI-1 gene.Therefore,this study focused on the pathogenesis of EVI-1 gene and AML,and explored the traditional chemotherapy drug arsenic trioxide(ATO)in EVI-1 transgenic zebrafish and in vitro THP-1,K562 leukemia cell lines to EVI-The mechanism of action 1 and its influence on downstream signaling pathways provide a theoretical basis for ATO clinical treatment of EVI-1 positive leukemia or other solid tumors.Methods:The recombinant plasmid Tol2-GFP-EVI-1 was injected into the zebrafish embryo to construct Tg(Tol2-GFP-EVI-1)zebrafish line(TE zebrafish),and the embryo development of TE zebrafish was observed by microscope and survived.Statistics are made.Detection of hematopoietic markers(including GATA-1,runx-1,mpo,lmo2,pu.1,scl,GATA-2,and c-myb)in TE fish and WT control fish embryos by RT-q PCR.The smears of the peripheral blood of TE and WT zebrafish were observed by Wright-Giemsa staining.TE zebrafish and WT zebrafish were sequenced by transcriptome sequencing(RNA-seq)and GO,KEGG analysis and RT-q PCR and western blot techniques were used to verify the sequencing results.When TE zebrafish develops to 16 hpf,50?M ATO is added to 3dpf,TE zebrafish and TE zebrafish are sequenced by transcriptome sequencing(RNA-seq)and GO,KEGG analysis and RT-q PCR and western blot techniques are used.The sequencing results were verified.RT-q PCR and western blot were used to detect the expression of EVI-1 in the newly diagnosed leukemia patients and 5 leukemia cell lines,and THP-1 and K562 cells were screened for subsequent experiments.Transfecting EVI-1 gene small interfering RNA(si RNA-EVI-1)and negative control interference fragment(si RNA-control)results into THP-1 and K562 cells and knocking down EVI-1 gene expression,using CCK8,cells Cyclic flow technique was used to detect the change of tumor cell proliferation level;cell smear HE staining and apoptosis flow technique were used to detect the change of tumor cell apoptosis level.Detection of NRAS after EVI-1 knockdown by western blot.The effect of the ERK pathway(total ERK,P-ERK,p27 kip1)and the role of EVI pathway inhibitor U0126 in demonstrating the effect of EVI-1 on the proliferation of tumor cells by regulating the NRAS/ERK pathway.Using the targetscan(Prediction of mi RNA targets)database to predict the mi RNAs that bind to the NRAS gene,select let-7,mi R196,mi R326,mi R505,mi R124,mi R361 as the target mi RNA,and detect the EVI-1 and predicted by RT-q PCR.The relationship between the target mi RNAs and the expression level of mi R124 was negatively correlated with EVI-1 gene.The dual luciferase reporter assay was used to detect that mi R124 directly binds to the 3'UTR of the NRAS gene and inhibits its expression.Using cell cycle flow technique and CCK8 technology to detect the tumor suppressor gene mi R124 regulates the proliferation of tumor cells by inhibiting the expression of NRAS and ERK pathway.Western blot analysis of JNK pathway and downstream apoptosis-related factors(total JNK,P-JNK,PUMA,P-p53,PUMA,Bcl-2,Bcl-xl,Bax,caspase 3,caspase)after EVI-1 knockdown 9)The impact.THP-1,K562 leukemia cell line was added with 0 ?M,1 ?M,3 ?M ATO by RT-q PCR,western blot,CCK8,cell smear HE staining and flow cytometry to detect ATO in vitro leukemia cell line EVI Inhibition of the-1 gene,regulation of downstream ERK and JNK pathways,and effects on tumor cell proliferation and apoptosis levels.Results:The results of this experiment constructed Tg(Tol2-GFP-EVI-1)transgenic zebrafish fish line,and found that the survival rate of TE zebrafish was significantly lower and there were different degrees of embryonic multiple organ development abnormalities(small eyes,curved body,pericardial edema).And no tail,etc.)phenotype.TE zebrafish has embryonic hematopoietic defects,and EVI-1 gene is overexpressed in the early hematopoietic process of zebrafish.Myeloid and erythroid hematopoietic markers(GATA-1,runx-1,mpo,lmo2,pu.1,scl,GATA-The expression dysregulation of 2 and c-myb)and the accumulation of hematopoietic stem cells in the peripheral blood circulation of adult fish are similar to the key phenotypes of human AML.Compared with WT control,TE zebrafish has 788 differentially expressed genes,and various signaling pathways related to embryonic hematopoiesis and development are abnormal.The abnormal activation of MAPK pathway is the most characteristic change.ATO can inhibit the expression of EVI-1 gene in TE zebrafish,and can reverse the activation of MAPK pathway in zebrafish due to overexpression of EVI-1 gene.The expression of EVI-1 gene was increased in primary leukemia cells and leukemia cell lines,and the expression level of EVI-1 was highest in THP-1 and K562 cells.By knocking down the EVI-1 gene in THP-1 and K562 cells,the proliferation of tumor cells was significantly inhibited,and the level of apoptosis was significantly enhanced.EVI-1 gene can inhibit the expression of tumor suppressor gene mi R124 in in vitro leukemia cells,indirectly promote NRAS expression and lead to abnormal activation of ERK pathway,promote cell G1/S phase transition and enhance tumor cell proliferation;EVI-1 gene in vitro leukemia.The cells can inhibit the apoptosis level of tumor cells by inhibiting the JNK pathway and its downstream apoptosis-related regulatory factors.ATO can inhibit the expression of EVI-1 in primary leukemia cells and THP-1,K562 leukemia cell lines,and inhibit the proliferation of tumor cells by inhibiting the expression of ERK pathway and related regulatory factors.At the same time,it can also activate the JNK pathway and its downstream apoptosis-related protein expression to promote the apoptosis level of tumor cells.Conclusions:In vivo,overexpression of EVI-1 gene can reduce the survival rate of transgenic zebrafish and cause dysplasia and hematopoietic disorder in zebrafish embryos,and activate MAPK pathway in zebrafish.ATO can inhibit the expression of EVI-1 gene in EVI-1 transgenic zebrafish and reverse the abnormal activation of MAPK pathway.In vitro,overexpression of EVI-1 gene in leukemia cell lines can inhibit the expression of mi R124 and indirectly promote the NRAS/ERK pathway and promote the proliferation of tumor cells.The EVI-1 gene inhibits the JNK pathway and inhibits the level of apoptosis in tumor cells.ATO inhibits the abnormally activated ERK pathway and promotes the JNK pathway in vitro by inhibiting the expression of EVI-1 gene and inhibiting tumor cell proliferation and inducing tumor cell apoptosis.