The Role Of E2A In ATPR-induced Cell Differentiation And Cycle Arrest In Acute Myeloid Leukemia Cells

Acute myeloid leukemia（Acute myeloid leukemia,AML）is a highly aggressive hematological malignancy.Its main characteristics are excessive proliferation,disorder of apoptosis,and stagnation of leukemia cell differentiation.The most common treatment methods have severe side effects,strong drug resistance,poor tolerance,and poor overall patient prognosis,thus,novel therapeutic approaches are needed.Our previous studies showed that 4-Amino-2-Trifluoromethyl-Phenyl Retinoic（ATPR）,a new derivative of all-trans retinoic acid（ATRA）,which is based on ATRA synthesized by structural modification.In vitro drug efficacy studies have found that ATPR can induce acute promyelocytic leukemia（APL）cell differentiation and cycle arrest,and it also effective for other AML subtypes cells of non-APL.Previous studies found that ATPR has the advantages of long drug effect,high solubility,low toxicity,from ATPR compared with ATRA.In vitro anti-tumor activity test results show that ATPR has obvious therapeutic effects on leukemia,lung adenocarcinoma,liver cancer,human gastric cancer,breast cancer and other tumor cells.Our group studies demonstrated its anticancer mechanism through the effect on enzyme activity,protein interaction,non-coding RNA downstream regulation etc.However many other mechanisms need to be further explored.As a member of the basic helix-loop-helix transcription factors（BHLH）,transcription factor E2A also known as transcription Factor-3（TCF3）,is located in the cytoplasm and regulates genes related to cell proliferation and differentiation.The E2A gene encodes the proteins E12 and E47.Its BHLH domain mediates the dimer and binds to the E-box within DNA,and the Ad1/Ad2 domains recruit p300 and GCN5 to enhance the activation of target genes.E2A is also known as a regulator of early B cell development.E2A has been reported to be abnormally expressed in many human cancers and is closely related to cell differentiation,proliferation,metastasis,and poor prognosis.The E2A gene is a common target of chromosomal translocation in leukemia and can easily to produce fusion proteins,such as E2A-PBX1,and E2A-HLF,but the mechanism of its regulation in leukemia has not been clarified.Taken together,these results suggest that alteration of the E2A gene may play a key role in the pathogenesis of leukemia.Preliminary experimental results show that ATPR can significantly inhibit the expression of E2A protein and m RNA levels in AML cells.Therefore,this article will further clarify the role of E2A in ATPR-induced differentiation and cycle arrest of AML cells and explore possible molecular mechanisms to provide a certain theoretical basis for the preclinical research of the new drug ATPR.Objective:This experiment mainly explores the role of E2A gene in ATPR-induced differentiation and cycle arrest of AML cells and related mechanisms.Methods:1.The expression of E2A gene in AML cells after ATPR treatmentWestern blot was used to determine the expression of E2A gene in healthy participants,blood samples from AML patients,and different AML cell lines and normal controls.We cultured AML cell lines NB4 and thp-1 cells in vitro,the E2A protein and m RNA levels at different concentrations of ATPR and at different times were detected by western blot and q-PCR.Western blotting was used to detect the change in the expression level of E2A gene after treat of ATPR at the optimal concentration and time(10^-6M,72 h),compared with the positive control drug ATRA(10^-5 M,72 h).2.Effect of knockout or overexpression of E2A gene on differentiation and cycle arrest of AML cells induced by ATPRWe transfected AML cells with sh-E2A lentivirus to reduce E2A expression activity or LV-h-E2A lentivirus to increase E2A expression level,and ATPR(10^-6 M)was administration 3 days.For each experimental treatment group,the protein expression levels of cell surface differentiation antigens CD11b and CD14 were checked by Western blot and Flow Cytometry.Wright-Giemsa staining to observe the changes of cell morphology,and NBT reduction experiment to calculate the percentage of positive cell differentiation.The expression of cell cycle related proteins was detected by Western blot,and FCM detects the cell cycle G0/G1 phase,S phase,G2phase distribution.3.Analyze the roel of E2A gene in AML by transcriptome gene sequencing（RNA-seq）We collected the cells of the experimental group and sent them to the sequencing laboratory for subsequent experimental steps.4.The effect of E2A gene on the expression of c-Myc and P-53 in AML cells treated by ATPRThe expression of c-Myc and P-53 genes were determined with Western blot in blood samples from healthy participants,AML patients,as well as different AML cell lines and normal controls.Detect the effect of E2A and c-Myc gene in AML cells by co-immunoprecipitation（Co-IP）.The immunofluorescence double staining technique was used to observe the distribution of E2A and C-Myc genes in AML cells.The expression changes of c-Myc and P-53 genes in AML cells with knockout or overexpression of E2A gene were detected by western blot.Western blotting was used to detect the protein expression levels of c-Myc and P-53 genes at the optimal concentration and time(10^-6 M,72 h)of ATPR,compare with the positive control drug ATRA(10^-5 M,72 h).5.The effect of E2A gene knockout on tumors in vivoTo construct a mouse model of NSG xenograft tumor in vivo.Firstly,the effect of down-regulation of E2A gene expression on tumor growth was observed in vivo,and then the expression levels of cell differentiation and cycle arrest related proteins in mouse subcutaneous tumor tissues were detected by Western blot and immunohistochemistry staining.Results:1.The E2A gene is highly expressed in AML patients and their cell lines compared tonormal controls.In AML cell lines NB4 and thp-1 cells,the protein and m RNAexpression levels of E2A gene were inhibited by ATPR with a time-andconcentration-dependent manner.ATPR and ATRA has inhibitory effect on E2Agene activity in AML cells,compared with the control.2.The decreased expression activity of E2A gene can enhance the effect of ATPR induced AML cell differentiation and cycle arrest,while overexpression of E2A gene reverses the therapeutic effect of ATPR on AML.3.Based on the sequencing results,we established an E2A gene co-expression network through bioinformatics predictions and microarray results,and found that the key downstream factor for the differential expression of E2A gene is the c-Myc gene,and closely related pathway is the P-53 signal pathway.4.The silencing of E2A gene can inhibit the expression of c-Myc gene and activate the P-53 signal pathway,while overexpression of E2A reverses the above phenomenon.The E2A gene and c-Myc gene are co-localized in the nucleus of AML cells,and there is an interaction between the two.ATPR inhibits the E2A/c-Myc axis and activates the P53 signalling pathway。5.The results showed that compared with the control group,the tumours in the sh-E2A group developed sluggishly and the tumour volume and average tumour weight were significantly reduced.The expression level of cell differentiation proteins was increased,and the expression of cell cycle proteins was inhibited,which was consistent with the results of in vitro cell experiments.Conclusion:ATPR could downregulate the expression of E2A with treatment of AML cell,inhibit the downstream target gene c-Myc,and induce AML cell differentiation and cycle arrest via the P53 signaling pathway.