Functional And Molecular Mechamism Study Of Lysine Acetyltransferase 2A And CDK4/CDK6 In Pediatric Acute Lenkemia

(Ⅰ)Functional and molecular mechamism study of lysine acetyltransferase 2A in pediatric acute leukemiaObjective: Histone regulation and histone modification,main content of histone code,are becoming hot spots of cancer research.Based on this theoretical guidance and large sample screening with our independent designand quantitative PCR chips,we found that KAT2 A is one of histone modification genes which are significantly upregulated in pediatric acute leukemia.Due to the abnormal expression of KAT2 A in pediatric acute leukemia and its effect on the leukemia cells’ proliferation,apoptosis and so on have not been reported,so this study has obvious innovation.Methods: The expression profiles of histone modified genes in 30 children with ALL and 27 children with AML were detected by using self-designed histone modified gene quantitative PCR chip.The expression of KAT2 A in 105 children with AML and the relationship with clinical parameters were analyzed.Annexin-V assay kit was used to analyze cell apoptosis.Apoptosis-related gene quantitative PCR chips were used to detect the gene profile after transient interference with KAT2 A.IPA(Ingenuity Pathway Analysis)analysis was performed on these differentially expressed genes to find important signal pathways,Cells were subjected to histone modification analysis by knocking out the expression of KAT2 A in AML cells using a gene knockout system.Results: Based on this theoretical guidance and large sample screening with our independent designand quantitative PCR chips,we found that KAT2 A is one of histone modification genes which are significantly upregulated in pediatric acute leukemia.Early discovery: Oncomine database analysis showed KAT2 A is significantly upregulated in leukemia and other tumors.We examined the relationship between the expression of AML in clinical samples of 105 cases of children with KAT2 A and various clinical parameters.The results showed that KAT2 A expression in children with AML was significantly higher than that in control non leukemia children bone marrow samples(P<0.01).Further data analysis showed that the survival time of children with high expression of KAT2 A was significantly shorter than that of low expression children(P<0.01),median survival time was 23.7 months and 41.8 months,respectively.Clinical multivariate regression analysis showed that KAT2 A expression could be used as an independent prognostic risk factor.Functional studies of KAT2 A in leukemia cells will be analyzed through gene silencing system in vitro.Preliminary study of KAT2 A gene in leukemia cells play the function of cell apoptosis was analyzed using the Annexin-V test kit,results show that the instantaneous interference of KAT2 A gene can significantly improve the apoptosis rate of HL-60 and NB4 cells.These results suggest that KAT2 A gene may play a role in the apoptosis of HL-60 cells.Molecular mechanisms of KAT2 A in leukemia will also be developed.To elucidate the molecular mechanism of KAT2 A gene in the occurrence and development of leukemia,Detection of apoptosis related gene KAT2 A found a transient disturbance after 24 genes were up-regulated and 37 genes were significantly down regulated by IPA,the genes of these differences(Ingenuity Pathway Analysis)analysis,established the gene network interaction model,sorting out the candidate signaling pathway and its key genes.IPA analysis showed that the regulation of KAT2 A gene on apoptosis,proliferation and other genes are likely to be realized through 5 key signaling pathways,namely: NF-kappa B,MYC,P53,TNF and NR3C1.Using gene knockout system to knockdown KAT2 A expression in AML cells after analysis of histone modifications on these cells,experimental results show that the acetylation of histone H3 and K14 K9 were significantly decreased,K14 and K9 may modify the substrate KAT2 A.The functional mechanism of KAT2 A in AML may be related to the acetylation modification of H3K14 and K9,which lays an important foundation for subsequent studies.Conclusions: This study identified the clinical significance of upregulated expression of KAT2 A gene in pediatric acute leukemia,multivariate regression analysis showed that KAT2 A was an independent prognostic risk factor for childhood AML.KAT2 A regulated apoptosis of AML cells may be related to the acetylation of histone H3K14 and K9,which will provide an important basis for further research.(Ⅱ)Molecular mechanism of G1 arrest and cellular senescence induced by LEE011,a novel CDK4/CDK6 inhibitor,in leukemia cellsObjective: Overexpression of cyclin D1 dependent kinases 4 and 6(CDK4/6)is a common feature of many human cancers including leukemia.LEE011 is a novel inhibitor of both CDK4 and 6.To date,the molecular function of LEE011 in leukemia remains unclear.Methods: Leukemia cell growth and apoptosis following LEE011 treatment was assessed through CCK-8 and annexin V/propidium iodide staining assays.Cell senescence was assessed by β-galactosidase staining and p16INK4 a expression analysis.Gene expression profiles of LEE011 treated HL-60 cells were investigated using an Arraystar Human Lnc RNA Array.Gene ontology(GO)and KEGG pathway analysis were then used to analyze the differentially expressed genes from the cluster analysis.Results: Our studies demonstrated that LEE011 inhibited proliferation of leukemia cells and could induce apoptosis.Hoechst 33342 staining analysis showed DNA fragmentation and distortion of nuclear structures following LEE011 treatment.Cell cycle analysis showed LEE011 significantly induced cell cycle G1 arrest in seven of eight acute leukemia cells lines,the exception being THP-1 cells.β-Galactosidase staining analysis and p16INK4 a expression analysis showed that LEE011 treatment can induce cell senescence of leukemia cells.Lnc RNA microarray analysis showed 2083 differentially expressed m RNAs and 3224 differentially expressed Lnc RNAs in LEE011-treated HL-60 cells compared with controls.Molecular function analysis showed that LEE011 induced senescence in leukemia cells partially through downregulation of the transcriptional expression of MYBL2.Conclusions: We demonstrate for the first time that LEE011 treatment results in inhibition of cell proliferation and induction of G1 arrest and cellular senescence in leukemia cells.Lnc RNA microarray analysis showed differentially expressed m RNAs and Lnc RNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induces cellular senescence partially through downregulation of the expression of MYBL2.These results may open new lines of investigation regarding the molecular mechanism of LEE011 induced cellular senescence.