Study On The Mechanisms Of Inhibit Proliferation And Invasion Of Leukemia Cells By S Component Of Staphylococcus Aureus Via Methyltransferase SET8

Objective Acute myeloid leukemia(AML)is the most common type of leukemia in adults with high mortality rate,which seriously threatens human life and health.In previous studies,we reported that Luk S-PV can promote the apoptosis of AML cell line HL-60 and THP-1,reflected an anti-leukemia effect.Whether the epigenetic modification plays a role in it is unclear.Lysine methyltransferase SET8 is highly expressed in a variety of tumor cells and promotes the proliferation and invasion of tumor cells.However,the role of SET8 in AML has been rarely studied in the formation and development of AML.Therefore,the purpose of this study was to investigate the role of SET8 in AML and to explore whether Luks-PV exerts antileukemic effects by down-regulating SET8.Methods(1)To investigate the expression of set8 mRNA in peripheral blood leukocytes of clinical patients,RNA was extracted and quantified by real-time quantitative polymerase chain reaction(qRT-PCR)to detect the expression of set8 mRNA in the peripheral blood of primary AML patients and normal individuals.(2)To investigate whether Luks-PV downregulate SET8.We used different concentrations of Luks-PV(0,0.25,0.5,0.75,1.0μM)incubated with THP-1,Hl-60 cells 24 h,using qRT-PCR and western blot to detect SET8 mRNA levels and protein levels.Western blot was used to detect the monomethylation level of Histone H4 20 th lysine residue(H4K20me1)in Luks-PV group.(3)To investigate whether the down-regulation of lysine methylation transferase SET8 has anti leukemic effect,we constructed the SET8 inhibit-expression lentiviral vector,negative control lentiviral vector,and transfected it into AML cell line THP-1,HL-60 cells respectively.Fluorescence microscopy,qRT-PCR and western blot used to verify the effect of SET8 inhibition,screened by puromycin.The CCK8 assay,flow cytometry,Transwell assay,Western blot were used to assess the effection of SET8 inhibition on cell proliferation,apoptosis,invasion and H4K20me1 levels.Results(1)Compared with normal individuals,the expression levels of SET8 were increased significantly in primary AML patients(P<0.01).(2)After treatment with Luks-PV,the SET8 mRNA and protein levels of THP-1 and HL-60 cells were decreased in concentration-dependent manners(P<0.05);The H4K20me1 levels of THP-1 and HL-60 cells were both decreased after treatment with Luks-PV.(3)Flow cytometry results showed that compared with negative control,the apoptosis of THP-1 and HL-60 cells infected by SET8 si RNA were both increased significantly,(19.57±2.56% VS 6.98±0.95%)and(18.26±2.34% VS 4.9±0.33%)(P<0.01),respectivily.(4)The CCK8 assay suggested that the proliferation in THP-1 and HL-60 cells were significant inhibited by SET8 interference(P<0.05).(5)Down regulate of SET8 by target SiRNA inhibits THP-1 and HL-60 cells invasion and migration(P<0.05).(6)The H4K20me1 levels of THP-1 and HL-60 cells were both decreased after transfected with SET8 inhibit expression lentiviral vector.Conclusion(1)SiRNA-mediated partial depletion of SET8 reduced THP-1 and HL-60 cells proliferation,inhibited their invasion,and promoted apoptosis.(2)Luks-PV might exerts anti-leukemic effects by down regulate SET8-H4K20me1 level.