Comparative Study Of NUDT21’s Regulation Of RUNX1 Through APA Mechanism To Affect The Biological Properties Of MDS And AML Cell Lines

Objective:Myelodysplastic syndromes(MDS)are heterogeneous myeloid neoplasms that arise from the clonal expansion of mutant pluripotent stem cells.It is characterized by bone marrow failure,abnormal cell morphology,and ineffective hematopoiesis,and approximately 30%of patients will develop acute myeloid leukemia(AML).RUNX1,a transcription-associated regulator widely expressed in hematopoietic cells,plays a key role in regulating the development of human hematopoiesis and is the most common mutated transcription factor in MDS,present in approximately 15%of MDS patients,and is associated with poor prognosis,thrombocytopenia and increased risk of transformation to AML.Alternative polyadenylation(APA)is an important post-transcriptional regulatory mechanism involved in a variety of diseases,including cancer.Approximately 70%of mammalian genes contain variable polyadenylation sites and therefore can encode mRNAs with different 3’ UTRs.CFIm25 is a subbase of cleavage factor I encoded by NUDT21,which is required for RNA 3’ end cleavage and polyadenylation.In our previous study,NUDT21 was found to be involved in APA,and our group constructed NUDT21 knockdown plasmid shRNAl with puromycin and NUDT21 overexpression plasmid OE with puromycin,and in this study,we found that NUDT21 regulates post-transcriptional modifications affecting RUNX1 and influencing the biological properties of MDS and AML cell lines through sequencing.By constructing AML and MDS cell models,we aimed to further investigate the effects of NUDT21,RUNX1,and the biological properties of MDS and AML cells,and thus to speculate whether the APA mechanism involving NUDT21 plays a role in MDS and AML patients.Methods:1、Agarose gel electrophoresis for identification and endotoxin-free amplification of existing plasmid modelsThe plasmids were amplified by transforming the plasmids into DH5a,the amplified broth was densely coated in LB solid medium and the monoclonal colonies were picked,extracted by alkaline lysis,and subjected to agarose gel electrophoresis.The plasmids were identified and the endotoxin-free plasmids were obtained by the endotoxin-free microextraction kit and used to construct cell models.2、Construction of AML,MDS cell models with NUDT21 knockdown and overexpression using a lentiviral transduction systemThe expression plasmids and packaging plasmids were transfected into 293T cells using liposome transfection.48h and 72h lentiviral supernatants were collected and infected with AML cell lines K562,THP-1,HL-60,and MDS cell lines MUTZ-1 and SKM-1.puro was used to screen the cells successfully infected with plasmids and qPCR and Western bolt were used to identify the expression levels of NUDT21 in each line.The expression levels of NUDT21 in the cell membrane phenotype were determined by qPCR and Western bolt.The successful knockdown cell models were named K562-NUDT21-shRNA1,THP-1-NUDT21-shRNA1,HL-60-NUDT21-shRNA1,MUTZ-1-NUDT21-shRNA1,SKM-1-NUDT21-shRNA1,and the overexpression models were K562-NUDT21-OE,THP-1-NUDT21-OE,SKM-1-NUDT21-OE.3、Transcriptome sequencing to detect the relationship between NUDT21 and APA in cellular models and downstream targetsThe cDNA libraries were sequenced on the Illumina HiSeq X Ten platform.Differential expression analysis was performed using the DESeq(2012)R package.P<0.05,FoldChange>2,or FoldChange<0.5 were used as differential expression thresholds.The differentially expressed genes were analyzed by systematic clustering,and the differentially expressed genes were analyzed by the GO enrichment method and KEGG pathway enrichment method,respectively.After resequencing by StringTie,the reference genome and known annotated genes were compared using CuffCompare software for gene structure extension and new transcript identification.Analysis of selective splicing of differentially regulated transcripts,isoforms,or exons using ASProfile.Transcriptome sequencing by Shanghai Ouyi Biomedical Technology Co.4、Q-PCR and Western bolt to verify the relationship between NUDT21 and RUNX1 in sequencing dataThe sequencing data showed a correlation between NUDT21 and RUNX1.Q-PCR and Western bolt were used to verify the relationship between NUDT21 and RUNX1 in the naked cell group,knockdown model group,and overexpression model group.5、CCK-8 assay to detect proliferation in each group of cell modelsK562-NUDT21-shRNA1,THP-1-NUDT21-shRNA1,HL-60-NUDT21-shRNA1,MUTZ-1-NUDT21-shRNA1,K562-NUDT21-OE,THP-1-NUDT21-OE were cultured in 1640 medium containing puro;SKM-1-NUDT21-shRNA1,SKM-1-NUDT21-OE were cultured in IMDM medium containing puro.The absorbance was measured at 0h,24h,48h,72h and 96h,and three sets of replicate wells were set up at each time point and the experiment was repeated three times.The results were calculated from the standard curve and compared with the proliferation of cells in each group.6、Flow cytometry for the cycle,apoptosis,proliferation,and cytokines in each cell modelIn order to detect more indicators of the cell models,we labeled the cell models with propidium iodide and RNaseA and performed flow cytometry to analyze the cycle;we labeled the cell models with Annexin and PI and performed flow cytometry to analyze the apoptosis;we used the kit to detect and analyze the cytokines of the cell models;we used the flow antibodies CD117-PC5 and CDllb-PE to label the cell models and performed flow cytometry to analyze the proliferation and differentiation of the cell models.7、Intracellular localization of NUDT21 and RUNX1 interactions by immunofluorescenceTo observe the intracellular distribution of NUDT21 and PIGN,cells were made into cell smears and fixed in permeation closure,incubated with antibodies against NUDT21 and PIGN,then conjugated with coupled fluorescent secondary antibodies,and then stained with DAPI on the nuclei of the cells.The co-localization of the green fluorescent NUDT21 protein with the red fluorescent PIGN protein and the blue fluorescent nuclei in each group of cells was observed using laser confocal microscopy.Results:1、The completed MDS and AML cell models were screened and identified using qPCR and Western bolt.indicated that the model was successfully constructed.2、mRNAs from THP-1,SKM-1,and MUTZ-1 cells were sequenced using next generation sequencing technology and next-generation the data were analyzed by Dapars(Dynamic analysis of alternative polyadenylation by RNA sequencing)method.The results showed that the RUNX1 3’UTR was shortened after the knockdown of NUDT21 and vice versa for the overexpression of NUDT21.3、CCK-8 analysis of cell proliferation in each group at 96h showed that NUDT21 knockdown inhibited the proliferation of K562(P<0.05),THP-1(P<0.001)and MUTZ-1 cells(P<0.01)and increased the proliferation of SKM-1 cells(P<0.001),but had no significant effect on HL-60 cells(P>0.05).4、CD11b and CD117 expression was significantly increased in K562-shRNA1(P<0.05,P<0.001)and SKM-1-shRNA1(P<0.001,P<0.05)and decreased in TIP-1-shRNA1(P<0.05,P<0.01).5、In overexpressed cells,the G0/G1 phase was shortened in AML cell lines K562(P<0.05)and THP-1(P<0.01)and increased in MDS cell lines(P<0.001).In knockdown cells,G0/G1 phase increased in AML cell line K562(P<0.001)and THP-1(P<0.01),whileG0/G1 phase remained increased in MDS cell line(P<0.001).6、Knockdown of NUDT21 led to a significant increase in apoptosis in AML cell lines K562(P<0.001)and THP-1(P<0.001),while overexpression of NUDT21 led to a decrease in apoptosis in AML cell lines K562(P<0.01)and THP-1 cells(P<0.001);in MDS cell lines,knockdown of NUDT21 led to a significant increased(P<0.001),while overexpression of NUDT21 also led to increased apoptosis(P<0.01),but the increase was smaller.7、It was found by immunofluorescence assay that NUDT21 was expressed centrally in the nucleus,while the fluorescence intensity of RUNX1 was reduced after knockdown,again centrally in the nucleus.8、IL-4 increased in AML cell lines and decreased in MDS cell lines;IL-10 increased in AML cell lines and decreased in MDS cell lines;IL-17 increased in AML cell lines and decreased in MDS cell lines;and IFN-γ was generally elevated after knockdown of NUDT21.Conclusion:This study suggests that NUDT21 is involved in regulating the biological functions of MDS and AML cell lines through the APA mechanism.Meanwhile,NUDT21 promotes apoptosis and inhibits proliferation of MDS cells through the APA mechanism,blocks MDS cells in the G1 phase,and decreases the expression of IL-4,IL-10,and IL-17 in MDS cells;inhibits apoptosis and promotes proliferation of AML cells,shortens the G1 phase of AML cells,and increases the expression of IL-4,IL-10 and IL-17 in AML cells to promote the transformation of MDS to AML.