Effect Of The Interaction Of 5-aza-2'-deoxycytidine And TNF-α On The Expression Of IER3 Gene And The Proliferation,Apoptosis And Differentiation Of Acute Promyelocytic Leukemia Cell Line NB4

Objective To measure methylation level of IER3 gene in the cell lines 0f nine malignant hematonosis by Methylation Specific PCR(MSP) and DNA sequencing,and then the screened hypermethylated cell line was used to as the subject of this study ;To discuss how 5-Aza-2'-CdR would reverse hypermethylation of IER3 and influence the expression of IER3 ;To discuss the interaction of 5-Aza-2'-CdR and TNF-αon the expression of IER3 and proliferation,apoptosis,differentiation of NB4 cell.Methods Methylation Specific PCR(MSP) and DNA sequencing was used to measure methylation level of IER3 gene in the cell lines of nine malignant hematonosis; Annexin V FITC/PI was used to estimate the apoptosis of NB4 cell;MSP was used to measure methylation level of IER3 gene under the action of drugs;The mRNA expression level of IER3,DNMT1,DNMT3A,DNMT3B was detected by quantiative reverse transcription-polymerase chain reaction(qRT - PCR);western-biotting was used to estimate the protein level of IER3,DNMT1,DNMT3A,DNMT3B;MTT method were used to evaluate the effect of 5-Aza-2'-CdR as well as interacting with TNF-αon the survival of NB4 cell ;Annexin V FITC/PI was used to detect the apoptosis,cell cycle and differentiation antigen CD11b,CD14 of NB4 cell after 48h disposal of 5-Aza-2'-CdR or/and TNF-α. Results⑴Hypermethylation of IER3 was present in nine malignant hematopoiesis cell lines.⑵The methylation level of IER3 was attenuated afer 48h disposal of 5-Aza-2'-CdR,but the effect was not obvious.⑶qRT-PCR tests showed that the expression of DNMT1,DNMT3A,DNMT3B was down-regulated after 48h of disposal of 5-Aza-2'-CdR .Although 5-Aza- 2'-CdR and TNF-αcould up-regulate the expression of IER3 mRNA respectively,the effect of interaction of 5-Aza-2'-CdR and TNF-αwas more apparent.⑷The results of western-blotting showed us that 5-Aza-2'-CdR could inhibit expression of DNMTs in protein level.Although 5-Aza- 2'-CdR and TNF-αcould up-regulate the expression of IER3 protein respectively ,the effect of interaction of 5-Aza-2'-CdR and TNF-αwas more apparent.⑸The MTT tests showed us that 5-Aza-2'-CdR and TNF-αcould inhibit the growth of NB4 cell after 48h,72h of disposal of drugs respectively.The inhibition of NB4 cell between 5-Aza-2'-CdR and interaction after 48h disposal had no significant differences,but it had obvious differences after 72h.The difference between treated group and untreated group had statistical significance after 48h,72h,but it had no statistical significance between the experimental group 2 and 5 after 48h,the same to 3 and 6;it had statistical significance no matter which group.⑹The Annexin V FITC/PI tests showed us that the apoptosis of NB4 after 48 disposal of interaction of 5-Aza-2'-CdR and TNF-αwas more apparent than single action;The inhibition of cell cycle s after 48 disposal of interaction was more apparent than single action;The positive rate of CD11b,CD14 of NB4 after 48 disposal of interaction was higher than single action,it had statistical significance no matter which group.Conclusion⑴Abnormal methylation of IER3 gene exists widely in nine malignant hematopoiesis cell lines. ⑵5-Aza-2'-CdR could up-regulate the expression of IER3 through inhibition of DNMTs , especially the DNMT1.⑶The interaction of 5-Aza-2'-CdR and TNF-αcould up-regulate the expression of IER3 more obviously than single action.⑷The interaction of 5-Aza-2'-CdR and TNF-αcould significantly inhibit NB4 cell proliferation and reduce the cell cycle s,it also could promote NB4 cell apoptosis, differentiation.