The Function And Mechanism Of TRIM22 Targeting EIF4E In The Process Of Leukemia Cells Differentiation

Background: The interferon-inducible protein TRIM22(also known as Staf50(stimulated trans-acting factor of 50 kD),including the RBCC region,the B-box region and the coiled-coil region,is assigned to the three-domain protein TRIM family.TRIM22 is one of the target genes of tumor suppressor gene P53,and its overexpression can activate NF-κB.Meanwhile,and the N-terminal RING domain and the C-terminal SPRY domain are important for their mediating processes in NF-κB activity,antiviral and autoimmune diseases.It has been shown that TRIM22 with anti-tumor effect is involved in the inhibition of viral replication and has exhibits RING domain-dependent E3-ligase activity,but its specific molecular function has not been elucidated.Eukaryotic translation initiation factor 4E(eIF4E)is a cap-binding protein that specifically recognizes the 5’ end of the mRNA’s cap structure and initiates translation.Excessive eIF4 E expression can lead to increased proliferation,escape from apoptosis,tumor invasion and metastasis.In hematoma,eIF4E’s overexpression is more common both in lymphoma and myeloid tumors including myelodysplastic syndromes,chronic myeloid leukemia(CML)and acute myeloid leukemia(AML),especially M4,M5 subtype and CML patients with acute phase change.The detection of eIF4E’s expression is expected to be one of the methods of monitoring tumor molecular markers.The reverse change between TRIM22 and eIF4 E in the myeloid differentiation of acute myeloid leukemia cells induced by ATRA were observed in the early stage of our study.TRIM19 has been shown to rely on the RING domain to bind to eIF4 E to inhibit its activity.Thus,we speculate that TRIM22 with similar structure may also have a similar effect and can be expected to be a new target for acute myeloid leukemia treatment.Objective: In order to investigate the function of TRIM22 and the interaction with eIF4 E in the differentiation of human acute myeloid leukemia cells for further illustration the mechanism of TRIM22 targeting to regulate eIF4 E,and to provide an important basis for finding new targets for leukemia treatment.Methods: The model of HL-60 and NB4 cells differentiation induced by ATRA was established in vitro experiment.The TRIM22 and eIF4E’s variation of gene and protein expression were detected by using RT-PCR,q-PCR and Western Blotting.In addition,after adopting electroporation technology to depress or over-express TRIM22,the effect on cell function and protein expression level of eIF4 E was detected respectively by CCK-8,flow cytometry and Western Blotting.Finally,the interaction of TRIM22 and eIF4 E was verified by using CO-IP.Results: After induced by ATRA,TRIM22 gene and protein expression levels of HL-60 and NB4 cells were increasing,while the eIF4 E gene and protein expression levels were reducing.The mRNA relative expression level of TRIM22 was gradually increasing from 1.01±0.15 to 30.98±2.79(F=280.70,P=0.000)and the level of protein relative expression was gradually increasing from 0.22±0.03 to 0.51±0.05(F=51.43,P=0.000)after the ATRA induction for NB4 cells.However,the mRNA relative expression of eIF4 E was gradually decreasing from 1.01±0.09 to 0.47±0.06(F=20.52,P=0.000).With the same trend,the level of protein relative expression was gradually decreasing from 0.97±0.02 to 0.64±0.09(F=14.70,P=0.001).The expression level of PE-CD11 b of the TRIM22 over-expression group with ATRA detected by flow cytometry(78.8±2.0)% was higher than the transfection group of empty vetor with ATRA(58.7±2.7)%(t=9.54,P=0.000);Besides,the expression level of PE-CD11 b of the cotransfection group with ATRA(61.6±3.8)% was lower than the TRIM22 over-expression group with ATRA detected by flow cytometry(78.8±2.0)%(t=8.19,P=0.000).The proliferation inhibition rate(0.25 ± 0.01)% of NB4 cells in TRIM22 over-expression group was higher than that in empty vector group(0.03 ± 0.02)%(t=15.47,P=0.000).Meanwhile,the protein level of eIF4 E changed reversely after over-expressing the gene level of TRIM22(t=4.99,P=0.000).That TRIM22 acts by combining eIF4 E is verified by the CO-IP experiment.Conclusions: The mRNA and protein expression of TRIM22 were gradually increased during the differentiation of HL-60 and NB4 cells induced by ATRA,while the expression of eIF4 E mRNA and protein decreased gradually.TRIM22 inhibits cell proliferation and promotes cell differentiation during the process of NB4 cells differentiation induced by ATRA and plays an important role by targeting eIF4 E to inhibit its expression.