Effect Of Methylation Inhibitor Of 5-aza-CdR And As₂O₃ On The Expression Of SHP-1, JAK1 And JAK2 Of Leukemia Cellines

Objective: DNA methylation is the process that under the effect of DNA methylase,set S-adenosine-L-methionine(SAM) as methyl donor, transfer methyl onto a special basic group.The normal function of gene not only depends on a normal structure , but also depends on a normal state of methylation.The abnormality of DNA methylation takes an important part in the occurrence of tumor. We have found that the reason why activation of oncogenes and the inactivation of anti-oncogenes is that low methylation or high methylation of these genes. With the happening of tumor,we can often see that the level of methylation of the whole genome will fall, at the same time, the special oncogenes will usually stay as a state of low methylation, however, transcription inhibition induced by high methylation of promoter sequence of promoter region could make some anti-oncogenes inactive expect for gene mutation or gene deletion.It has been improved that the abnormality of DNA methylation in tumor cells relate to the abnormality of activity of DNA methyltransferases.So ,inhibition of the activity of DNA methyltransferases could return the expression of methyltransferases, so it disturb the occurrence and development of tumor.And methylation inhibitor of 5-aza-CdR and As₂O₃ can conform into DNA,then interfere the ability of the acceptance to methyl,or directly restrain the activity of DNA methyltransferases,finally it can synthesize low methylation DNA.The study was to investigate the expression of SHP-1,JAK1 and JAK2 of leukemia cells, meanwhile further understood the influence of methylation inhibitor to the expression of genes and the possible mechanisms of SHP-1 in JAK/STAT and leukemia.Methods: U937 and K562 cells were cultured in RPMI 1640 and treated with different concentrations of 5-aza-CdR and As₂O₃. The cells were collected at different time points, the real-time quantitative PCR was used to detect the mRNA expression level of SHP-1,JAK1 and JAK2 gene in trial teams and control teams.Results:1 Real-time quantitative PCR results showed when U937 was treated with 5-aza-CdR and As₂O₃ respectively or together,with the extension of time,the expression of SHP-1mRNA was increased, the expression of JAK1mRNA and JAK2mRNA was decreased.2 When U937 was treated with 5-aza-CdR and As₂O₃ respectively or together, with the elevation of the drugs concentration,the expression of SHP-1mRNA was increased, the expression of JAK1mRNA and JAK2mRNA was decreased. 3 When K562 was treated with 5-aza-CdR and As₂O₃ respectively or together,with the extension of time,the expression of SHP-1mRNA was increased, the expression of JAK1mRNA and JAK2mRNA was decreased.4 When K562 was treated with 5-aza-CdR and As₂O₃ respectively or together, with the elevation of the drugs concentration,the expression of SHP-1mRNA was increased, the expression of JAK1mRNA and JAK2mRNA was decreased.5 When U937 and K562 were treated with 5-aza-CdR or As₂O₃,the expression of SHP-1mRNA was lower,the expression of JAK1mRNA and JAK2mRNA was higher than were treated with 5-aza-CdR and As₂O₃ together in low concentrations after 24h.6 There was a negative correlation between the expression of SHP-1mRNA and JAK1mRNA(Linear correlation and regression analysis, r=-0.8400 , P<0.01).There was a negative correlation between the expression of SHP-1mRNA and JAK2mRNA(Linear correlation and regression analysis, r=-0.9068,P<0.01).Conclusions:The expression of SHP-1 could be increased ,JAK1 and JAK2 could be degraded in U937and K562 when treated with 5-aza-CdR and As₂O₃.Meanwhile they were presented time-dependent and dose-dependent. The two drugs belong to methylation inhibitor ,they had a synergism in the three genes. We thought that cooperation with the two drugs enhanced the demethylation. So the expression of SHP-1 which is anti- oncogenes would be increased.In conclusion,SHP-1 possibly come into effect by inhibiting JAK/STAT,then tumor would be inhibited.It might provide a particular approach to leukaemia therapy .