The Function And Mechanism Of EIF2S3 Gene In T-Cell Acute Lymphoblastic Leukemia

Objectives:Eukaryotic translation initiation factor 2 subunit 3(EIF2S3)plays an important role in the initiation process of protein translation.Previous studies have revealed the expression level of EIF2S3 in patients with non-small cell lung cancer and colorectal cancer,suggesting that EIF2S3 is expected to be a biomarker and therapeutic target for cancer.The aim of this article is to investigate the expression level and biological function of EIF2S3 in T-cell acute lymphoblastic leukemia(T-ALL).Methods:Clinical samples were collected to verify the difference in EIF2S3 expression between ALL patients and normal people by quantitative fluorescence PCR(qRT-PCR).Western blot(WB)was used to detect the protein expression level of EIF2S3 in T-ALL cell lines.EIF2S3 overexpressing cell(MOLT4-EIF2S3 and JURKAT-EIF2S3)and knockdown cell(JURKAT-shRNAs)were constructed by molecular cloning.CCK-8assay,clone formation assay,flow cytometry and invasion assay were used to verify the effect of EIF2S3 on T-ALL.The underlying mechanism of EIF2S3 function was explored by high-throughput transcriptome sequencing and WB.Results:The results of qRT-PCR showed that the EIF2S3 mRNA level in patients with Tcell acute lymphoblastic leukemia was lower than that in normal controls(P<0.05).WB detection showed that EIF2S3 was overexpressed in JURKAT and HUT102 cells,and under-expressed in MOLT4,CCRF-CEM,A3 and HUT78 cells.The EIF2S3 protein levels of overexpressed cell lines(MOLT4-EIF2S3 and JURKAT-EIF2S3)were significantly higher than those of the control group,while the EIF2S3 protein levels of knockdown cell lines(JURKAT-shRNA1 and shRNA2)were lower than those of the control group.CCK-8 assay showed that overexpression of EIF2S3 inhibited the proliferation of MOLT4 and JURKAT cells(P<0.05),while knockdown of EIF2S3 promoted the proliferation of MOLT4 and JURKAT cells(P<0.05).Clone formation experiments showed that the number of clones formed was decreased in EIF2S3 overexpressing cell lines(P<0.05),and the opposite result was obtained in EIF2S3knockout(P<0.05).In cell cycle assay,the proportion of MOLT4-EIF2S3 and JURKAT-EIF2S3 cells in G0/1 phase was increased compared with the control group(P<0.05),while the number of cells in S phase was significantly decreased(P<0.05).High-throughput sequencing was performed on the two groups of EIF2S3 overexpression cell lines.Using P < 0.05 and FC(fold change)≥1.5 as the screening criteria,a total of 494 common differential genes were screened among which 292 genes were up-regulated and 202 genes were down-regulated.Functional enrichment analysis showed that up-regulated genes were mainly involved in cell apoptosis,cell proliferation,cell migration,cell adhesion,metabolism and so on,while downregulated genes were involved in immune response,DNA synthesis,leukocyte migration,protein phosphorylation,G0/1 phase transformation and so on.The pathway enrichment analysis showed that the major signaling pathways involved in the differential genes included p53 signaling pathway,PI3K-Akt signaling pathway,FOXO signaling pathway and PPAR signaling pathway.There were 23 differentially expressed genes in these four pathways,and the qRT-PCR result showed that 16 of them had statistically significant differences in the MOLT4-EIF2S3 and MOLT4-Vector cells(P<0.05).WB result showed that EIF2S3 inhibited the phosphorylation level of Akt protein(P<0.05),while no consistent trend was observed in other pathway proteins.Conclusions:(1)The expression of EIF2S3 was down-regulated in patients with T-cell acute lymphoblastic leukemia.(2)EIF2S3 inhibited the proliferation and clonal formation of T-ALL cells(MOLT4 and JURKAT cells),as well as leading to G0/1 cell cycle arrest.(3)EIF2S3 may exert its biological function through PI3K-Akt signaling pathway.