The Effects Of DNA Methylation On Expression Of ABO Gene In Leukemia

ObjectiveABO blood group is the most important blood group system in human.ABO blood group is determined by forward and reverse blood typing.Forward blood typing detect ABO antigens on the erythrocyte membrane,and reverse blood typing detect ABO antibodies in the serum.When forward and reverse blood typing were detected at the same time,ABO blood group typies could be confirmed.But some hematological malignancy or solid tumor patients may appeared the condition that forward and reverse blood typing were not corresponded.It was easy to make blood typing error,which was directly related to the safety of clinical blood transfusion.Alteration of ABO antigens in hematological malignancy was first reported by van Loghem et al who described very weak A antigen expression on the RBCs of an acute myeloid leukemia(AML)patient,who had previously shown normal A antigen expression.Possible mechanisms for inactivation of ABO include allelic loss(loss of heterozygosity,LOH),mutation and silencing by DNA methylation.At present,it has been found that the DNA methylation was closely related to ABO antigen decreased of hematological malignancy patients.In this study,we detected the expression intensity of ABO antigen and DNA methylation level of ABO promoter between hematological malignancy patients and normal control,then we compared the differences between each others,and in order to investigate the relevant favtors of weak ABO antigen,and the relationship between DNA methylation level of ABO promoter and weak ABO antigen of ematological malignancy patients.Furthermore,leukemia cell lines K562,HL-60 and Jurkat were cultured,and treated with demethylation drug Decitabine(DAC).Then the biological effects of three leukemia cell lines,ABO genotypes,expression level of ABO mRNA,methylation level of the CpG dinucleotides in ABO promoter were detected before or after added DAC,and the effects of DNA methylation of ABO promoter on the expression of ABO gene in vitro were discussed.Methods1.The ABO blood group was detected in 119 new diagnosed leukemia patients and 196 normal controls,then we observed the expression intensity of ABO antigen.Furthermore,we combined with the leukemia subtype,age,sex,laboratory test,risk level of leukemia patients,in order to find the relationships between weak ABO antigen and others factors.DNA methylation of ABO promoter of leukemia patients and normal controls were detected by bisulfite sequencing PCR(BSP),and the methylation level of ABO gene promoter were compared in leukemia and mormal control group.2.The leukemia cell lines K562.HL-60 and Jurkat were cultured and treated with different doses of decitabine.The proliferation of cells were examined by MTT assay,cell apoptosis were detected by flow cytometry Annexin V FITC/PI double-labeled,the migration and matrigel invasing tests were detected by transwell chanber.Then the growth inhibitory rates were counted and the optimal concentration and time were found.3 · The leukemia cell lines K562.HL-60 and Jurkat were treated with different doses of decitabine,ABO genotypes were detected by Polymerase chain reaction sequence-specific primer(PCR-SSP).ABO mRNA were detected by Quantitative Real-time PCR(RT-PCR),and DNA methylation level of ABO promoter were detected by Bisulfite sequencing PCR(BSP).Results1.There were 33 A-blood type cases,43B-blood type cases,39 O-blood type cases,4 AB-blood type cases in new diagnosed leukemia patients,what had no statistical difference from the normal group.9 cases were weak ABO antigen in new diagnosed leukemia patients and none in the normal group.There were no statistical differences in the distribution of ABO blood group,age,hepatosplenomegaly,lymphadenovarix,plt,precursor clusters from bone marrow,immunophenotyping,LDH,risk level between weak ABO antigen group and normal ABO blood antigen group.The pateins of weak ABO antigen group had higher percentage of male(77.8%&30%),lower WBC(32.26×1 O9/L&82.69x109/L)and Hb level(64.00g/L&85.94g/L)and higher DNA methylation level(18.91%&10.76%)than the normal ABO blood antigen group,p<0.05.The methylation rate of ABO promoter in AML and ALL was 52.68%and 12.05%respectively,which was obviously higher than those in control(2.01%).DNA methylation level in ALL were obviously higher than that in AML,P<0.05.The methylation rate of ABO promoter in chronic myeloid leukemia(CML)was 2.78%,which had no statistical difference from the normal group,P>0.05,but that in a case of CML blast crisis phase was 92.56%,which was obviously higher than that in chronic phase.2.After K562,HL-60,Jurkat cells were treated with 0.5?mol/L?1?mol/L?5?mol/L?10?mol/L?20?mol/L DAC for 24h,cells growth were significantly inhibited.The inhibition rates of expermental group were obviously higher than that of control group(0?mol/L DAC).DAC could inhibite the proliferation of three cell lines in dose-dependent manner and time-dependent manner.After concentration 1?mol/L?10?mol/L of DAC treated with three cells 72 hours,Annexin V FITC/PI double labeling showed that apoptotic rates were higher than the control group,P<0.05.There was statistically significant difference in the inhibition rate of invasiveness and migration ability after 1?mol/L?10?mol/L of DAC were treated with three leukemia cell lines 24 hours between the control group and expermental groups(P<0.05).3 ABO genotypies of K562,HL-60 and Jurkat were O1O1,O1A1 and A1O2.The brightness of bands in treated group and control group were no significant changes of K562 cell lines.The brightness of bands in treated group were lighter than control group in HL-60 and Jurkat cell lines.ABO mRNA expression of K562 treated group was 1275.67±35.86,which was obviously higher than that in HL-60 and Jurkat,P<0.05.The methylation rate of ABO promoter in HL-60 and Jurkat treated group were 58.14%and 96.74%,and the the expression of ABO mRNA were 0.5410.07 and 0.8210.16 respectively,After the treatment with decitabine,the methylation rate of ABO promoter were decreased and the expression of ABO mRNA were increased in a dose-dependent manner,which were obviously different from control group,P<0.05.Conclusion1.The cases of weak ABO antigen were frequently appeared in AML patients.Weak ABO antigen patients have higher DNA methylation level of ABO promoter,lower WBC and Hb level than the normal ABO antigen patients.The methylation level of ABO promoter in leukemia patients is obviously higher than that in normal group.2.DAC can obviously inhibit the cell survival,induce leukemia cells apoptosis and inhibit leukemia cells invasiveness and migrationwith time and dose dependent manners.It also can significantly down-regulate methylation level of ABO promoter and up-regulate expression of ABO mRNA in leukemia cell lines.3.Leukemia cell lines K562,HL-60 and Jurkat are all express ABO antigens on cell surface.The methylation rate of ABO promoter and the expression of ABO mRNA show a negative correlation.Gene silencing caused by DNA methylation was one of the important mechanism of ABO antigens decrease in acute leukemia.