Study On The Anti-erythroleukemia Mechanism Of Tetrandrine Base On FLI1-HDC-Mediated Inflammation Pathway

Objective:To explore the role of Friend virus-induced leukemia integration site 1（FLI1）mediating Histamine decarboxylase（HDC）and its downstream signaling pathways in erythroleukemia,and to study the anti-erythroleukemia action mechanism of Tetrandrine based on this pathway,to provide a new theoretical basis for the treatment of leukemia.Methods:（1）The effect of FLI1 gene（sh FLI1）for knockdown in HDC was analyzed by RNAseq and Real time-PCR;（2）Promoter analysis methods and chromatin immunoprecipitation（Ch Ip）experiments were used to verify the binding of FLI1 to the HDC promoter in leukemia cells;（3）The expression level of the lentivirus-mediated HDC（sh HDC）gene was determined by Western blotting and Real time-PCR;MTT analysis of the effects of supplementation with or without histamine on the proliferation of HEL cells lines and sh HDC cells;（4）The expression of related inflammatory genes in sh FLI1 cells was analyzed by RNAseq and Real time-PCR;（5）The effect of compound Tetrandrine on the viability and proliferation of HEL cells was detected using the MTT method;（6）Using the flow-cytometric technology,After detection of Tetrandrine on HEL cells,The expression of the cycle and apoptosis;（7）Analyzed the expression levels of FLI1 and HDC genes in compound Tetrandrine by Western blotting and Real time-PCR on HEL cells;Using the molecular docking method to verify the effect of Tetrandrine on FLI1 protein;（8）Using an F-Mu LV virus-based animal model,To assess the in vivo effects of compound Tetrandrine in leukemic mice.Results:（1）The RNAseq and Real time-PCR results showed that FLI1 and HDC expression were positively correlated.（2）Promoter and chromatin immunoprecipitation（Ch Ip）results confirm that FLI1 is transcriptionally regulated by direct binding to the HDC promoter.（3）Western blotting and Real time-PCR data indicated that HDC protein levels and m RNA levels were downregulated in sh HDC cells.MTT results showed that the proliferation of cells with or without histamine in HEL cells and sh HDC cells was not affected.（4）RNAseq analysis and Real time-PCR results showed that the inflammatory genes CXCR2 and IL1B were downregulated in sh HDC cells.（5）MTT results showed that compared with DMSO,Tetrandrine（1μM,3μM,5μM）inhibited（15.54±2.58）%,（21.08±3.54）%,（72.44±8.44）%,IC₅₀was 5μM,and Tetrandrine（1μM,3μM,5μM）inhibited HEL cell proliferation time-dose dependent.（6）Cell flow results showed that apoptosis increased in dose with increasing Tetrandrine concentration,and Tetrandrine blocked the cycle position S phase of HEL in leukemia cells.（7）Western blotting and Real time-PCR data indicate that Tetrandrine downregulates FLI1 and HDC expression at the protein and m RNA levels.Molecular docking results indicate that Tetrandrine and FLI1 proteins are combined.（8）Animal experimental data show that Tetrandrine significantly prolonged the survival time of leukemic mice.Conclusions:1、FLI1 binds to the HDC promoter in erythroleukemia cells,and HDC involved in histamine biosynthesis is a direct target of FLI1,and FLI1-induced HDC is involved in leukemia progression independently of the histamine synthesis pathway.2、HDC influences leukemogenesis through the tumor microenvironment,whereas HDC inhibition in vivo slows down leukemia progression.FLI1 participates in the pathogenesis progression of erythroleukemia through HDC regulating the related inflammatory genes CXCR2,IL1B.3、Compound Tetrandrine inhibited erythroleukemia HEL cell proliferation,induced apoptosis,arrested the cell cycle into S phase,and downregulated FLI1 and HDC expression,prolonging the survival of leukemic mice,thereby inhibiting leukemic progression.