A Preliminary Study Of The Effect And Mechanism Of DNMT3A-mutated Acute Myeloid Leukemia On Macrophage Polarized Phenotype

Objective: We sought to detect the effect and molecular mechanism of DNMT3 A mutated AML on the polarizing phenotype of macrophages compared to wild-type AML cells,and explore potential therapeutic targets.Methods: Using table lentivirus-mediated transfection to overexpress DNMT3 A R882H and clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)technology to knockout DNMT3 A in SKM-1 cell lines,the present study constructed R882 H SC1 and R882 H SC2 cell strain,and KO cell strain.Sanger Sequencing,RT-q PCR and Western Blot were employed to detected the expression of DNMT3 A and DNMT3 A R882H in m RNA and protein levels.CCK8 kit was used to detect the proliferation differences of the experimental group(R882H cells and KO cells)and the control group(SKM-1 cell group and NC cell group).Flow cytometry was used to detect the apoptosis differences of the above cell strains under different concentrations of cytarabine.Differentially expressed genes between wild-type SKM-1,KO cell strain and R882 H cell strains were identified by using RNA sequencing(RNA-seq).To find the the possible mechanisms,KEGG and Gene set enrichment analysis(GSEA)was used to execute pathway enrichment analysis.The above cells were co-cultured with THP-1 macrophages.Macrophages polarization phenotypes were examined by flow cytometry,ELISA and RT-q PCR.RT-q PCR was performed to measure the gene expression levels of proinflammatory cytokines in AML cells before and after co-cultivation.The expression levels of inflammatory cytokines in coculture supernatant were detected by ELISA.In addition,an animal model of xenograft tumor was established in 4-week old SCID mice.The proportion and location of M2 macrophage was determined utilizing immunofluorescence staining.Results: R882 H SC1 cell strain,R882 H SC2 cell strain,and KO cell strain were successfully constructed.RNA-seq found that KO cells and R882 H cells co-expressed 96 up-regulated genes and 185 down-regulated genes(such as JUN(Jun proto-oncogene),FOS(Fos proto-oncogene),CEBPB(CCAAT Enhancer Binding Protein Beta),KLF2(Kruppel Like Factor 2),IL-1β(interleukin-1β),MIP-1β/CCL4(Macrophage Inflammatory Protein 1β,including CCL4L1,CCL4L2)and CCL3L1,CCL3L3(proteolytic truncation of CCL3/MIP-1α).KEGG enrichment analyses showed that a large number of differential genes are enriched on inflammatory immune-related pathways,such as the TLR(Toll-like receptor)signaling pathway and the NF-κB(nuclear factor kappa B)signaling pathway.GSEA database showed a down-regulation of TLR signaling pathways in KO cell strain and R882 H cell strain(P<0.001).The STRING database shows that there are complex interactions between the differential proteins above.Among them,JUN and FOS are the key regulatory proteins and are located in the interaction center of these proteins.After cocultivation in vitro,the M1-related markers in co-cultured macrophages in the experimental group(R882H cells and KO cells)and OCI-AML3 cells group were significantly down-regulated compared with the control group(SKM-1 cell group and NC cell group)(P <0.05),while the M2-related markers were significantly up-regulated(P <0.01).In the above co-cultivation system,proinflammatory cytokine(MIP-1α、MIP-1β and IL-1β)in the AML cells in experimental group were significantly lower than those in the control group(P<0.001),while the immunosuppressive factors(IL-10 and TGF-β)were not significantly different from those in the control group(P>0.05).In cocultivated supernatant,the level of the pro-inflammatory cytokine in the experimental group were significantly lower than in the control group(P<0.05),but levels of the immunosuppressive factors were significantly higher than in control group(P<0.01).In xenograft experiments,the tumor volume and the proportion of tumor-infiltrating M2-like macrophages in the experimental group were significantly larger than those in control group.Conclusion: DNMT3A-mutated AML can reduce the expression of inflammatory factors MIP-1α,MIP-1β and IL-1β,regulate the polarization of leukemia-associated macrophages and thus enhance their own immune escape ability.