The Prognostic Significance Of The Expression Of Splicing Factor TRA2 And Its Mechanism In Acute Myeloid Leukemia

Objective:1.To explore the expression and prognostic significance of splicing factor TRA2in patients with acute myeloid leukemia（AML）,to provide references for finding new biomarkers and prognostic indicators for AML.2.To investigate the effects of TRA2 expression on the apoptosis,proliferation and cell cycle of acute myeloid leukemia cells（THP-1 and mv4-11）and preliminary mechanism.Methods:1.A total of 28 newly diagnosed patients with AML were selected as the De novo AML and collected clinical data,15 patients with non-hematological diseases and healthy volunteers were selected as the Control.Bone marrow was extracted to detect the expression of TRA2A and TRA2B m RNA and protein,and the relationship between TRA2B expression and clinical features and prognosis of De novo AML was analyzed.The prognostic significance was verified in the Cancer Genome Atlas（TCGA）.2.THP-1 and mv4-11 cells stably over-expressing and knocking-down TRA2A and TRA2B were generated using TRA2A and TRA2B over-express and knockdown lentivirus.The changes of cell apoptosis,proliferation,cell cycle and the expression of cell cycle-related proteins Cyclin D,Cyclin E,Cullin-3L,Cullin-3b were detected after verified the successful transfection of lentivirus,and then performed statistical analysis.Results:1.Compared with the control group,the expression of TRA2B m RNA in patients with de novo AML was significantly reduced,and the expression of protein was significantly increased（P<0.05）;while the expression of TRA2A m RNA and protein were no different from those in control（P>0.05）.2.The expression of TRA2B in de novo AML was not related to hepatosplenomegaly,degree of anemia,WBC,RBC,PLT,peripheral blood and bone marrow blasts,and was not related to the risk,fusion gene situation and chromosome situation of patients with de novo AML（P>0.05）.3.In FAB typing,the expression level of TRA2B in M2,M4 and M5 patients was significantly lower than that in M3（P<0.05）,and there was no difference in the expression of TRA2B between M3 and control（P>0.05）.There was no difference in the expression of TRA2B among M2,M4 and M5 patients（P>0.05）.4.Survival analysis showed that the overall survival rate（OS）was higher in patients with TRA2B high expression group compared with TRA2B low expression group（P<0.05）,whereas there was no significant difference in OS between TRA2A high and low expression groups in patients with AML（P>0.05）.5.More than 90%of cells in each lentivirus infection group expressed green or red fluorescence after TRA2 lentivirus transfected the THP-1 and mv4-11 cells.The verification results showed that after over-expression of TRA2A,the TRA2A m RNA expression in the over-expression group was higher than that of the over-expression control（P<0.05）,while the expression of TRA2A protein was not different from the over-expression control（P>0.05）.The expression of TRA2A m RNA and protein were lower than the knockdown control after TRA2A knockdown（P<0.05）.The verification results of TRA2B were consistent with TRA2A.6.Apoptosis results suggested that compared with over-expression control or knockdown control,there were no difference in the apoptosis rate and expression of apoptosis related protein caspase3 of over-expression or knockdown of TRA2 in THP-1 and mv4-11 cells（P>0.05）.7.The results of cell proliferation showed that the OD₄₅₀of THP-1 cells were greater and mv4-11 cells were lower than knockdown control after transfected TRA2A knockdown lentivirus.And the OD₄₅₀of THP-1 and mv4-11 cells were significantly lower than the knockdown control after transfected TRA2B knockdown lentivirus（P<0.05）.8.The cell cycle results showed that the percentage of THP-1 and mv4-11 cells in G1 phase was higher than that of the knockdown control（P<0.05）,and the percentage of G2 phase and proliferation index were lower than that of the knockdown control（P<0.05）,while the percentage of S phase had no difference with the knockdown control after transfected TRA2B knockdown lentivirus（P>0.05）.9.The expression of cell cycle protein Cyclin D,Cyclin E in THP-1 and mv4-11cells were significantly lower than those of knockdown control after transfected TRA2B knockdown lentivirus（P<0.05）.10.The expression of cell cycle-related protein Cullin-3L in THP-1 and mv4-11cells were significantly increased（P<0.05）,while the expression of Cullin-3b was significantly lower than those of knockdown control after transfected TRA2B knockdown lentivirus（P<0.05）.Conclusion:1.Low TRA2B m RNA and high TRA2B protein expression were observed in patients with AML,whereas the expression of TRA2A m RNA and protein were unchanged.2.The expression of TRA2B was associated with the prognosis of patients with AML,and patients with high TRA2B expression had a better prognosis.3.In AML cells,the knockdown of TRA2B may regulate the alternative splicing of Cullin-3,promote the expression of Cullin-3L,and then increase the ubiquitination degradation of Cyclin D and Cyclin E,block the cell cycle in G1 phase,and significantly inhibit the proliferation of AML cells.Overexpression and knockdown of TRA2 gene had no effect on the apoptosis of AML cells.