The Impact On DNA Methyltransferase Of Simvastatin In Acute Myeloid Leukemia Cell Line NB4

Backgroud:Acute myeloid leukemia is a heterogeneous disease of hematopoietic system malignant clonal proliferation. Abnormalities of the primitive cells and immature cells over proliferation in bone marrow, inhibit the normal hematopoietic function, and widely infiltration of liver, spleen and other organs. Clinical manifestations such as anemia, bleeding, infection and infiltration are caused by bone marrow failure and leukemia cell infiltration. The average survival of leukemia is short, and it is a serious threat to human health. With the rapid development of modern medical science, AML diagnosis and treatment have made great progress, but according to the US SEER cancer statistics review of the data shows no significant improvement in disease-free survival and overall survival.Epigenetics is the study of meiotically and mitotically heritable changes in gene expression that are not coded for in the underlying DNA sequence. DNA methylation is an important molecular mechanism of abnormal hematologic malignant disease, and is a common epigenetic changes in acute leukemia, in which methylation of tumor suppressor gene inactivation is most important. DNA methyltransferase enzyme catalyzed genomic DNA methylation,and they are involved in many biological processes such as embryonic development, gene expression and regulation. Overseas study found, DNA methyltransferase gene have abnormal expression in leukemia patients and leukemia cell lines, and with some of the tumor suppressor gene inactivation are closely related. Commonly used leukemia chemotherapy drugs have problems such as drug resistance and relapse, but methylation drugs have certain curative effect to resistance and relapse patients because of their different mechanism of action. The most commonly used decitabine,5-azacytidine, etc. demethylating drugs mostly through specific inhibition of the activity of DNMT function.Statins are mevalonate reductase inhibitors, in addition to regulating lipid metabolism effects, they also have anti-inflammatory, immunosuppressive, anti-platelet aggregation, inhibition of renal cell proliferation and anti-tumor effects. The standard dose of statins have good safety and tolerability. Studies confirmed that, treatment with lovastatin downregulates DNMT activity, leading to BMP2 promoter demethylation, and may be able to be used as differentiating agents in combined or adjuvant therapy in CRC with the CpG island methylator phenotype.In this study, we discussing the impact on DNA methyltransferase of simvastatin in acute myeloid leukemia cell line NB4 and its possible mechanism, to explore the new drugs for leukemia treatment.Objective:In acute myeloid leukemia cell line NB4, to study the impact on DNA methyltransferase of simvastatin and explore its possible mechanism.Methods:1. Experiments were divided into 4 SIM-treated groups:NB4 cells treated with different concentrations of simvastatin and with different times, the final concentrations are:0 μmol/L、5μmol/L,10 μmol/L and 15 μmol/L.2. DNMT activity/inhibition assay were used to detect the changes of DNA methyltransferase (DNMT) activities in NB4 after treated with simvastatin.3. Quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine the expression of DNMT1、DNMT3A and P53 mRNA.4. Western-blotting were used to analyze the expression of DNMT1-. DNMT3 A and DNMT3B protein levels.5. Annexin V/PI double staining detection were used to detect the changes in the rate of apoptosis after simvastatin treatment.Results:1. After simvastatin treatment, DNMT overall activity reduced (P<0.05), and showed a dose-dependent manner.2. After treatment with simvastatin, DNMT1, DNMT3A mRNA expression levels were significantly reduced, and P53 mRNA expression level increased (P<0.05)3. After simvastatin treatment, DNMT1, DNMT3A protein levels decreased (P 0.05), DNMT3B protein expression did not change (P> 0.05).4. Simvastatin promote NB4 cell apoptosis, and showed a certain amount of time, dose-dependent manner.Conclusion:1. Simvastatin in NB4 cells can reduce DNA methyltransferase activity, reduce the level of gene expression and protein expression levels, so as to achieve demethylation and tumor suppressor gene re-expression.2. Simvastatin can promote NB4 cell apoptosis.3. This study demonstrates that simvastatin is expected to become a new drug treatment of acute leukemia act as DNA-hypomethylating agents.