| Objective:N-myc downstream regulated gene-1(NDRG1)is an anti-oncogenic signal interferer that plays a critical role in various cancers.Previous studies have shown that NDRG1 promotes the differentiation of acute myeloid leukemia cells,but the relevant mechanisms have not been fully elucidated.The role of NDRG1 in acute T-cell lymphoblastic leukemia(T-ALL)remains unknown.Our previous study found that eukaryotic translation initiation factor 2 subunit 3(EIF2S3)has an inhibitory effect on the progression of acute lymphoblastic leukemia(ALL).In this study,we investigated the biological function and mechanism of NDRG1 binding with EIF2S3 in T-ALL,aiming to identify new therapeutic targets and provide a basis for disease prognosis evaluation.Methods:The expression level of N-myc downstream regulated gene-1(NDRG1)in T-ALL patients and normal T cells was analyzed using the GEO database,followed by real-time quantitative PCR(RT-qPCR)to detect NDRG1 expression levels in peripheral blood mononuclear cells of T-ALL patients and healthy individuals.The expression level of NDRG1 protein in T-ALL cell lines was detected by Western blot(WB).JURKAT and MOLT4 cells were selected to construct NDRG1-HA overexpression cell lines,and CCRF-CEA cells were selected to construct NDRG1 knockdown cell lines.Cell morphology was observed by Wright-Giemsa staining of the cells on slides.The biological function of NDRG1 in T-ALL cell lines was explored using CCK-8 proliferation assay,flow cytometry cell cycle analysis,and cell invasion assay.Differential gene analysis of JURKAT-NDRG1 and JURKAT-vector cell lines was conducted through high-throughput sequencing,and the biological information analysis of proteins that bind to NDRG1 was performed using the InAct database.Validation was conducted through immunoprecipitation(IP)experiments by transfecting NDRG1-HA expression plasmids into 293T cells.Western blot was used to detect the expression of NDRG1 protein in EIF2S3 overexpression and knockdown cells to explore the relationship between them.The effect of EIF2S3 on NDRG1 protein was investigated using a protein half-life experiment.Finally,the function and pathway enrichment of co-regulated genes of NDRG1 and EIF3S3 were analyzed.Results:Obtaining the expression matrix of NDRG1 by downloading the dataset of T-ALL patients from GEO,analysis of datasets GSE139613 and GSE141140 showed that the mRNA expression levels of NDRG1 in T-ALL patients were lower than those in normal T cells,with P values of P=0.0421 and P=0.0011,respectively.RT-qPCR analysis of T-ALL patient specimens suggested that NDRG1 expression was lower than that in normal individuals(P=0.0136).WB analysis of NDRG1 protein expression levels in T-ALL cell lines showed that JURKAT,A3,HUT102 and MOLT4 cell lines did not express or had low expression levels of NDRG1,while HUT78 and CCRF-CEM cell lines had high expression levels of NDRG1,with the expression in CCRF-CEM cells being the highest.The expression levels of JURKAT-NDRG1-HA and MOLT-NDRG1-HA cell lines overexpressing NDRG1 and their corresponding control groups showed that the exogenous tagged protein HA was significantly expressed,and the expression levels of NDRG1 were significantly higher than those in the control group.Knockdown of NDRG1 in CCRF-CEM cell lines suggested that the expression of NDRG1-shRNAl and NDRG1-shRNA2 decreased compared to the control group,while the knockdown effect of NDRG1-shRNA3 cells was not significant.JURKAT-NDRG1-HA,MOLT-NDRG1-HA,CCRF-NDRG1-shRNA1,CCRF-NDRG1-shRNA2,and their corresponding control cell lines were selected for cell function experiments.Observing the morphology of NDRG1-overexpressing and knockdown cell lines under an OLYMPUS microscope,compared with the control group,the NDRG1-overexpressing cell lines showed a decrease in cytoplasmic vacuoles,coagulated chromatin in the nucleus,and no visible nucleoli,while the control group showed loosely packed chromatin in the nucleus and faintly visible nucleoli.The knockdown of NDRG1 in the cells showed enlarged cell bodies,irregular nuclear shapes with petal-like folding and twisting,and deeply stained cytoplasm.The CCK-8 proliferation assay showed that overexpression of NDRG1 inhibited the proliferation of JURKAT cells(P<0.05),while knockdown of NDRG1 promoted the proliferation of CCRF-CEM cells(P<0.05).In the flow cytometry cell cycle assay,the proportion of cells in the G0/1 phase and pre-DNA synthesis phase(G0/1)increased in JURKAT-NDRG1 and MOLT4-NDRG1 cells compared to the control group(P=0.000334、P=0.000002),while the proportion of cells in the DNA synthesis phase(S phase)decreased(P=0.000366、P=0.003704);the proportion of cells in the G0/1(P=0.000007、P=0.000018)and S phases(P=0.000602、P=0.000051)decreased in NDRG1-shRNA1 and NDRG1-shRNA2 cells compared to the control group,while the proportion of cells in the post-DNA synthesis phase(G2/M phase)(P=0.000059、P=0.000020)significantly increased compared to the control group.In the cell invasion assay,the invasive ability of JURKAT-NDRG1(P=0.0003)and MOLT4-NDRG1(P=0.0155)cells was weakened compared to the control group,while the invasive ability of CCRF-shRNA1(P=0.0268)and CCRF-shRNA2(P=0.0285)cells with NDRG1 knockdown was enhanced compared to the control group.Analysis on the IntAct database website revealed that the NDRG1 protein binds to the EIF2S3 protein.A combined analysis of high-throughput sequencing data from JURKAT-EIF2S3 and JURKAT-NDRG1 was performed,with a threshold of FC(fold change)>1.5 and P<0.05.A total of 144 commonly upregulated genes and 77 commonly downregulated genes were identified,and the 221 regulated genes were subjected to bioinformatics analysis.Functional enrichment analysis(GO analysis)indicated that these genes were mainly involved in biological processes,biological regulation,cellular components,organelle components,transcription regulation,translation regulation,and T lymphocyte differentiation.Pathway enrichment analysis(KEGG analysis)revealed that the genes were mainly enriched in the RAP1 signaling pathway,PI3K-AktX signaling pathway,RAS signaling pathway,and P53 signaling pathway,with a total of 21 differentially expressed genes involved in these pathways.Thirteen differentially expressed genes from the RAS/PI3K-Akt signaling pathways were selected for RT-qPCR validation in JURKAT-EIF2S3 and JURKAT-vector,JURKAT-NDRG1 and JURKAT-vector groups,and the results showed that the expression levels of 12 genes were consistent with the sequencing results in JURKAT-EIF2S3 and JURKAT-vector groups,with 10 differentially expressed genes showing statistical significance.In the JURKAT-NDRG1 and JURKAT-vector groups,the results showed that the expression levels of 10 genes were consistent with the sequencing results,among which 7 differentially expressed genes were statistically significant.Western blot validation of core proteins in the RAS/PI3K-Akt signaling pathway indicated that the expression levels of RAS,pAkt,and pmTOR proteins in JURKAT-EIF2S3 and JURKAT-NDRG1 were lower than those in the control group.Drug sensitivity analysis showed that cells with low expression of EIF2S3 were more sensitive to drugs such as methylprednisolone,cyclophosphamide and carmustin,while cells with high expression of EIF2S3 were more sensitive to the drug ibrutinib.Cells with low expression of NDRG1 were more sensitive to drugs such as fostamatinib,LY-294002,and CDK inhibitors,while cells with high expression of NDRG1 were more sensitive to drugs such as imatinib and tic10.Conclusions:1.The expression of NDRG1 is down-regulated in T-ALL.2.Down regulation of NDRG1 expression increased the atypia of microscopic cell morphology and promoted cell invasion ability in acute T lymphoblastic leukemia cell line CCRF-CEM;Meanwhile,enables cells to progress from G0/1 and S phase into G2/M phase and promotes the proliferation ability of cells.3.NDRG1 can inhibit the invasive ability of acute T lymphoblastic leukemia cell lines JURKAT and MOLT4;Meanwhile,the proliferation ability of JURKAT cells was inhibited by arresting the cells in G0/1 phase.4.NDRG1 recruits EIF2S3 to increase protein stability.5.Common regulatory genes of NDRG1 and EIF2S3 inhibit the proliferation and cell cycle arrest of T-ALL cells by negatively regulating the Ras/PI3K-Akt signaling pathway. |
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