Effect Of The Interaction Of3-deazaneplanocin A And5-aza-2’-deoxycytidine On The Expression Of MST1Gene And The Proliferation、Apoptosis And Differentiation Of Acute Promyelocytic Leukemia NB4Cell Line

Objective：To detect the expression of MST1in leukemia patients andmalignant hematopoietic cell lines and investigate its clinical significance，then fiterthe malignant hematopoietic cell lines with low expression of MST1and taked it assubject to research; To measure methylation status of MST1gene in acutepromyelocytic leukemia NB4cell line by Methylation Specific PCR(MSP); Todiscuss the interaction of DZNep and5-Aza-2’-CdR on the expression of MST1andto detecte proliferation、apoptosis、differentiation of NB4cell.Methods：The expression of MST1was detected and its clinical significancewas analysised in clinical samples and5species of malignant hematopoietic cell lineswith quantitative real-time polymerase chain reaction and Western-blottingmethod,then NB4cell line was fitered with low expression of MST1;The methylationstatus of MST1gene in NB4cell line was measured by Methylation Specific PCR;The CCK-8assay methods was used to evaluate the effect on situation of cellproliferation in NB4cells after disposal of DZNep and5-Aza-2’-CdR; Hoechst33258fluorescent staining method were used to observe the situation of cell apoptosis,Apoptosis ratio was detected by flow cytometry with Annexin V，FITC/PI; real-timePCR were applied to identify the mRNA expression lever of MST1、Bcl-2、DNMT3A、DNMT3B and DNMT1，and Western blotting were applied to identifythe protein expression lever of MST1、 procaspase-3、procaspase-9. Results：(1)The expression of MST1in acute leukemia untreated patients wassignificantly decreased (P<0.05), With the improvement of the treatment of thedisease, the difference between complete remission in patients and normal personswas not statistically significant (P>0.05), For relapsed/refractory acute leukemiapatients, the expression of MST1was significantly lower than normal (P <0.05),at thesame time, the difference was not statistically significant with untreated patients (P>0.05); NB4cell line was fitered with low expression of MST1.(2) Hypermethylationof MST1was present in NB4cell line.(3) CCK-8assay demonstrated that DZNep and5-Aza-2’-CdR could inhibit the proliferation of NB4cells and a dose-dependent,which also inhibit G1-S conversion (P <0.05) the inhibition of was more apparentafter combination therapy(P <0.05); NB4cells after disposal with DZNep and5-Aza-2’-CdR were detected that apoptosis increased and nuclear stain were dense byHoechst33258fluorescence staining; and its apoptosis ratio increased by flowcytometry(P <0.05), the rate of apoptosis increased more obvious after combinationtherapy(P <0.05); they may also reduce the expression of Bcl-2and promote theactivation of procaspase-3and procaspase-9, promoting apoptosis accordingly.(4)DZNep may throught down the expression lever of DNMT3B and histonemethylation modification to upregulation the expression of MST1; and5-Aza-2’-CdRmainly through down the expression of DNMT3A; two-drug combination theexpressionof MST1raised more obvious.Conclusion:(1) The low expression of MST1in leukemia has the function oftumor suppressor genes,and with the process of treatment, the expression level ofMST1dynamic change.(2) Hypermethylation of MST1was present in NB4cell line.(3)DZNep and5-Aza-2’-CdR, to enable the cells arrest at the G1/S phase leaving thedifferentiation blocked, increased apoptosis, the combination is more obvious.(4)DZNep may throught down the expression lever of DNMT3B and histonemethylation modification to upregulation the expression of MST1; and5-Aza-2’-CdRmainly through down the expression of DNMT3A; two-drug combination theexpressionof MST1raised more obvious.