Study On Prognosis-related Alternative Splicing Events Of Acute Myeloid Leukemia And Cell Biological Function Of BCL2L11-γ Spliceosome

Objective:Acute myeloid leukemia(AML)is a kind of hematological malignant disease,and the prognosis of relapsed/refractory acute myeloid leukemia(RAML)is poor.It is very important to find out the molecular mechanism and further to discover the potential therapeutic targets of these diseases.The abnormality of alternative splicing(AS)is closely related to the prognosis of AML.In this study,bioinformatics was used to analyze TCGA-AML data.RNA splicing pattern,AML expression profile and relevant clinical information were obtained.Alternative splicing network was constructed,alternative splicing factors related to survival and genes involved in alternative splicing events were identified,and the correlation between alternative splicing events and the occurrence and prognosis of AML were analyed.The expression level of prognosis-related splicing factor,SRSF1,was tested in clinical samples,and the cell biology function experiment of SRSF1 and its target gene BCL2L11 was carried out to clarify the molecular mechanism of RAML pathogenesis and lay a theoretical foundation for exploring potential therapeutic targets of RAML.Methods:1.To obtain and integrate AS data of AML in TCGA database through bioinformaticsFirst of all,the data of the patients are obtained from TCGA and TCGASplice Seq databases,and then the data were screened and analyzed according to the criteria such as Percentage of Samples with PSI Value > 75%,clinical OS > 30 and so on;Secondly,the patients were divided into groups according to whether NMP1 was mutated or not,and R 3.5.1 software was used to identify and analyzed the abnormal AS events of the two groups of patients;Then survival-related AS events and AS events independent prognostic-related genes were identified through Cox regression analysis;Meanwhile,David tool was used to analyze the GO enrichment of prognostic genes,and the gene interaction network map was constructed by Cytoscape3.6.1;Finally,a prognostic model of AS was constructed and splicing factors related to the prognosis of AML were found.2.The expression level of SRSF1 in bone marrow of RAML patientsBone marrow samples of 30 AML patients with CR/PR and 30 RAML patients were collected.Total RNA was extracted from bone marrow samples by Trizol method and the expression level of SRSF1 in the two groups was detected by real-time quantitative PCR(q PCR).3.Effects of SRSF1 and different spliceosomes of BCL2L11 on cell proliferation and apoptosisTheexpression vectors of SRSF1,BCL2L11-γ spliceosome and BCL2L11-L spliceosome were constructed and transferred into cells using Lipofectamine 2000;After overexpression of SRSF1,cell proliferation was detected by CCK8 and apoptosis was detected by flow cytometry;RT-PCR was used to detect the expression levels of BCL2L11-L and γ splicesome after SRSF1 overexpression;Finally,CCK 8,high content imaging system and flow cytometry assay were used to examine the influences of different spliceosomes of BCL2L11 on cell proliferation and apoptosis.Results:1.A total of 129 AML patients were selected from the TCGA and TCGA Spliceseq database in this study.the AS events were analyzed,and the universality of AS events and the main event type is exon skipping(ES)in AML;We identified 239 prognostic genes such as BCL2L11,CASP3,and so on;Multiple splicing prognostic models were constructed and suggested that the group with higher incidence of AS had a poorer prognosis;The prognostic factors such as SRSF1 and RBM4 were defined;Among them,high expression of SRSF1 or low expression of RBM4 could predict poor prognosis of AML.2.The expression level of SRSF1 was higher in the RAML group than that of CR or PR AML group.(P<0.05).3.Overexpression of SRSF1 promoted the formation of BCL2L11-γ spliceosome in 293 T cells(P<0.05),and inhibited the apoptosis of cells.CCK 8 assay and apoptosis experiment showed that compared with the control group,there was no significant difference in cell proliferation and apoptosis after overexpression of BCL2L11-γ spliceosome(P>0.05),while cell proliferation decreased significantly(P<0.05)and apoptosis increased(P<0.05)after overexpression of BCL2L11-L spliceosome.Conclusion:1.There are seven alternative splicing events in acute myeloid leukemia,of which ES is the most common event.The genes involved are mainly related to biological processes such as cell cycle,mitotic checkpoints,and DNA integrity checkpoints.Alternative splicing events are associated with the prognosis of AML.High expression of splicing factor SRSF1 and low expression of RBM4 are related to poor prognosis.2.SRSF1 is high expression in patients with relapsed and refractory acute myeloid leukemia.3.BCL2L11 is one of the important targets of SRSF1.Overexpression of SRSF1 causes abnormal alternative splicing of BCL2L11,promotes the formation of BCL2L11-γ splicesome.BCL2L11-γ splicesome may play a role in AML drug resistance and progression by inhibiting apoptosis.