| Background:Acute myeloid leukemia(AML)is the most common type of adult leukemia.It is a clonal disease due to malignant proliferation of hematopoietic stem cells/progenitor cells.Retrospective studies based on global data show that there are about 1 million cases of AML each year,resulting in 147000 deaths.In China,acute myeloid leukemia is also one of the blood diseases with high incidence,which is a serious threat to people’s health and safety.Although some progress has been made in the study of AML,its specific pathogenesis is still inconclusive.Therefore,finding the potential mechanism related to the occurrence,development and prognosis of AML is still the focus of academic research in the field of hematological oncology.Objectives:1.Use bioinformatics to understand the expression of RNA binding protein NUDT21 in patients with acute myeloid leukemia(AML).2.Use RT-qPCR to determine the expression of RNA binding protein NUDT21 in patients with acute myeloid leukemia,and analyze its clinical significance related to the disease.3.To explore the effect of RNA binding protein NUDT21 on the biological function of acute myeloid leukemia cell line THP-1.Methods:1.Use the TCGA database online analysis tool UALCAN to analyze the expression of NUDT21 in different subgroups(FAB typing,gender,age,race,etc.)and correlate it with high-risk factors such as PML/RARα fusion gene,RAS activation and FLT3 gene mutation analysis.2.Using the GEO database,download the AML data set file(GSE12417)containing survival information(163 patients),and use the GEPIA online website to analyze the prognostic effect of NUDT21 expression in the TCGA database.3.Use cBioPortal and LinksedOmics to analyze the AML-related information in the TCGA data set to obtain NUDT21 co-expressed genes,and set the screening condition as the absolute value of Spearman’s correlation coefficient>0.5.Use the Venn diagram online tool to obtain the intersection gene set.4.Using WebGestalt(http://www.webgestalt.org/),perform GO function enrichment analysis and KEGG pathway enrichment analysis of NUDT21 expression-related genes.5.Collect 45 cases of peripheral blood samples from healthy subjects and 38 cases of peripheral blood samples from patients with acute myeloid leukemia,isolate PBMC(mononuclear cells),extract total RNA,and reverse transcribed into cDNA.Real-time polymerase chain reaction(real-time polymerase chain reaction,real-time PCR)method was used to detect the expression of RNA-binding protein NUDT21 in the two groups of studies,and RNA was explored at multiple levels and at multiple levels based on different clinical indicators The relationship between the expression level of binding protein NUDT21 and AML.6.Construction of shRNA expression plasmid PLKO.1-U6-PUROshNUDT21(abbreviated as shNUDT21)targeted to NUDT21:shNUDT21 infects THP-1 cells,Western Blot detection and RT-q PCR detection of shNUDT21 versus THP-1 Inhibition of cell NUDT21 protein and mRNA expression.7.After inhibiting the expression of NUDT21,use CCK8 to detect cell proliferation ability;use flow cytometry to detect cell apoptosis and cycle ability;use RT-qPCR and flow cytometry to detect cell differentiation ability.Results:1.Among the subtypes of AML FAB classification,the expression of NUDT21 in M0～M2 is higher than that of M3～M7,and compared with other age groups,the expression level of 61～80 age group is significantly higher than that of 81～100 age group(P<0.05).Secondly,there was no difference in the expression of NUDT21 in different gender,race,FLT3 mutation status,PML/RAR fusion status,and RAS activation status.2.GEO and TCGA database analysis both show that patients with high NUDT21 expression have a poor prognosis and a lower overall survival rate,but there is no statistical significance.(P>0.05)3.Use cBioPortal and LinkedOmics to obtain two NUDT21 expression-related gene sets;use the Wien method to unify the two gene sets to obtain 323 genes.4.GO analysis of NUDT21 expression-related genes showed that in terms of biological processes,it is mainly related to transcription regulation,DNA templated,cell apoptosis,DNA repair,cell apoptosis,protein transport,etc.;in terms of molecular functions,it is mainly related to Protein binding,nucleic acid binding,ATP binding,DNA binding,etc.are related;in terms of cell components,it mainly involves cell membrane,cytoplasm,and cell nucleus.KEGG analysis showed that NUDT21 expression-related genes were mainly enriched in lysosomes,endocytosis,Epstein-Barr virus infection,AMPK signaling pathway,cell cycle,cell differentiation and other processes.5.NUDT21 is highly expressed in AML patients,but there is no statistical difference between the age,gender,FAB type,peripheral blood white blood cell count,hemoglobin content,platelet count,and bone marrow primitive naive cells of patients in the NUDT21 high and low expression groups.6.The peripheral blood white blood cell counts of AML patients and normal people are not statistically significant.Normal human hemoglobin(142g/L[119g/L～171g/L])VS AML patient hemoglobin(76g/L[30g/L～154g/L])and normal platelet count(220×109/L[107×109/L～283×109/L])VS AML patient platelets(63×109/L[1×109/L～264×109/L])The counts were statistically significant(P<0.001).7.shNUDT21 was successfully constructed.Western Blot and Real-time PCR results showed that shNUDT21 can effectively inhibit the expression of NUDT21 protein and mRNA in THP-1 cells.8.After using shNUDT21 to inhibit the expression of NUDT21 in the THP-1 cell line,the proliferation ability of THP-1 cells was significantly reduced,apoptosis and differentiation were significantly increased,and there was no significant change in cycle.Conclusion:1.The expression of RNA binding protein NUDT21 is significantly increased in patients with acute myeloid leukemia.2.After the expression of RNA binding protein NUDT21 is inhibited,it can inhibit the proliferation of leukemia cells THP-1 and enhance cell apoptosis and differentiation.Regulating the expression of NUDT21 may become a new potential treatment strategy for acute myeloid leukemia. |
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