Research On BRD4in The Directional Differentiation Of Leukemia Cells And Its Role In The Key Mechanism

BackgroundSodium valproate (VPA) is a kind of traditional medicine in the treatment of epilepsy, has confirmed that it has histone acetylation in clinical dose range (HDAC) characteristic effects of inhibitors. It can improve the level of histone acetylation of histone and DNA, which combined with enhanced, so as to promote the transcription of target genes, gene expression regulation to correct abnormalities, finally can induce differentiation and apoptosis of tumor cells. In vitro, it can inhibit the growth of leukemic cells. At present, there are a large number of experiments show that histone deacetylase inhibitors (HDACi) on behalf of the drug VPA can significantly promote the all-trans retinoic acid (ATRA) on differentiation induction of leukemia cell line HL-60.BRD (a double bromodomain-containing protein bromodomain) is a member of the BET family, containing two bromo structure protein, can advantage with acetylated chromatin, which changes the chromatin conformation. Studies have found that, BRD4plays a critical role in the process of DNA replication, transcription, cell cycle regulation, tumor and virus genome separation. For the first time in2011September reported in the foreign literature, BRD4as a nucleotide regulatory target for the treatment of acute myeloid leukemia, shRNA or small molecule inhibitor of BRD4in vitro and in vivo can induce stable against leukemia, including terminal differentiation and leukemia bone marrow stem cell elimination. But in differentiation induction of leukemia treatment, the expression of BRD4, targeted expression of BRD4in the regulation of differentiation of the role is not very clear. This research through the differentiation of leukemia cell model of all-trans retinoic acid combined with HDAC inhibitor VPA induced, to explore different differentiation agent on the effect of changes in expression of BRD4in HL-60and the effect of leukemia cell lines, so as to explore the target of the anticancer mechanism, and for targeting combined application drugs in the treatment of acute promyelocytic leukemia, to achieve a better therapeutic effect and provide experimental basis.ObjectiveIn this experiment, human promyelocytic leukemia cell line HL-60as the research object, focus on the effect of all-trans retinoic acid (ATRA) combined with sodium valproate (VPA) molecular mechanisms regulating the expression of BRD4differentiation of HL-60cells induced, clarify BRD4mediated differentiation of leukemia cells mechanism and structural basis, open up new field for the study of cell differentiation, provide ideas and experimental new clues for leukemia treatment.MethodsModel ATRA and VPA to induce differentiation of leukemia cell line HL-60, to observe the morphology of HL-60cells by Wright-Gimesa staining, cell proliferation was measured by MTT assay, the expression of cell surface differentiation antigen CD11b was detected by flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), protein imprinting (Western blot) expression of BRD4from HL-60cells the level of mRNA and protein induced by the drugs24h,48h,72h. On the other hand, the construction of eukaryotic expression vector of BRD4, for the future lay the foundation by overexpression of BRD4, BRD4expression by siRNA interference method. Measured by x±s, were compared with t test, compared with single factor analysis of variance,p<0.05was statistically significant,p<0.01was significant difference.ResultsApplication of ATRA or VPA alone could significantly inhibit the growth of leukemia cells, inducing cell differentiation, under the microscope can be observed in HL-60cells to differentiate into mature myeloid cells, differentiation antigen cell surface expression, cell proliferation was inhibited, cell proliferation effect more obvious differentiation model, cell surface differentiation antigen expression increased more significantly induced by VPA lmmol/L+ATRA0.5μmol/L, both have statistical significance. Decreases in mRNA expression and protein level of BRD4decreased more obviously, differentiation model induced by the combination of VPA lmmol/L+ATRA0.5μmol/L, both have statistical significance. Further studies are required to determine its specific molecular mechanisms.ConclusionsATRA or VPA HL-60leukemia cells, inhibit cell proliferation, induce cells to differentiation, and the two drugs combined induced differentiation effect is better, indicating that VPA could promote the differentiation of HL-60cells induced by ATRA. The degree of BRD4expression with the degree of differentiation of acute promyelocytic leukemia HL-60cells was decreased, the transcription level and protein expression level of consistency, the malignant degree and leukemia is related, prompts in the pathogenesis of leukemia in inhibition of differentiation of BRD4cells, play an important role in the process of induced differentiation of acute promyelocytic leukemia cell leukemia. This study provides experimental and theoretical basis for the differentiation of myeloid leukemia in clinical treatment of VPA combined with small dose of ATRA, and provides a new idea for targeted therapy and medicine therapy for malignant tumor.