The Studies On Mechanisms Of 5-Aza In Regulating Proliferation,Differentiation And Migration Of Mononuclear Leukemia Cells

Background and Objective:5- Aza(5- nitrogen impurity cytosine nucleoside) is a kind of methylation inhibitors, 5-Aza has been approved to apply in clinical therapy of myelodysplasia(MDS) and acute myeloid leukemia(AML), and it inhibits cell proliferation and induces cell differentiation by demethylating various genes, including tumor suppressor genes, transcriptional factors, cell cycle inhibitor genes, etc. Although it acquires better effects in clinical application, drug resistance after 5-Aza treatment still appears. Cell migration and invasion based on 5-Aza treatment were considered as the key factors of drug resistance, however, little is known about the detailed mechanism.This suggests we use 5-Aza, demethylating drugs for leukemia treatment,the treatment method disadvantage.This project aims to study the impact 5-Aza THP-1in human acute monocytic leukemia cell migration proliferation, differentiation,possible mechanisms and explore effective programmes to address the negative impacts.Method:1.RT-PCR detection CCL2 and CCR2 expression,Transwell experimental observation of cell migration,Westernblot experimental detection of CCR2,ERK, p-ERK and AKT, p- AKT expression, ELISA method to detect the content of the CCL2.2.Flow cytometry instrument method to detect the change of CD14.Wright-Giemsa staining observe changes in cell morphology.3 Cells treated with 5 aza at different concentrations and different time,then take the CCK8 and Brd U experiments.Result:1.the expression of CCL2 and CCR2 are increased by 5Aza treated.5-Aza significantly promoted THP-1 cell migration;CCR2 inhibitor completely inhibits5-Aza-induced THP-1 cell migration compared with control group,ERK inhibitor can inhibit 5-Aza-induced THP-1 cell migration,CCL2 level of cell culture increased2.6-fold in the presence of 5-Aza compared with control group,the expression of p-ERK,CCR2 are increased by 5Aza treated.2.the level of CD14 expression significantly increased in 5-Aza-treated cells compared with that of DMSO-treated cells cell morphology of 5-Aza-treated cells was observed under inverted microscope and observed by Giemsa staining.3.cell growth was estimated by CCK8 stainin, Brd U staining results showed 5-Aza significantly inhibits THP-1 cell proliferation.Conclusion:5- Aza cause CCL2, CCR2 gene expression,Cause P- ERK expression;5-Aza through CCL2 CCR2- ERK signaling pathway activation THP 1 cells migration,5- Aza promote Cell differentiation and inhibition of THP 1 cells proliferation.