Loss Of C-Myc Suppressed The Expression Of CDK1 And Cyclin B1 Via The Targeted Inhibition Of Histone H₄ Acetylation And Induced G₂/M Arrest

Oncogene c-Myc that was located on chromosome 8q24.12-8q24.13,is the one of very important members of the Myc family.The proto-oncogene product c-Myc,a basic helix-loop-helix/leucine zipper?b HLH/LZ?–type transcription factor,is a master regulator of cell proliferation.c-Myc forms a heterodimeric complex with the smaller b HLH/LZ protein Max,and the c-Myc-Max complex binds to a specific DNA sequence?CACGTG?known as the E-box motif.This motif is located in the promoter region.In many human malignant tumors,the expression level of c-Myc is increased as a result of amplification or mutation of the c-MYC gene.Over-expression of c-Myc is involved in cell proliferation and apoptosis in the process of B-lineage acute lymphoblastic leukemia?B-ALL?formation,but the mechanism is not well understood.Here we established a c-Myc-knockdown model?Raji-KD?using Raji cells,and found that c-Myc regulates G₂/M-associated gene?CDK1 and cyclin B1?expressions by modulating TIP60/MOF-mediated histone H₄ acetylation?Ac H₄?,which could then be completely restored by re-introduction of cMyc gene to Raji-KD cells.The expressions of CDK1 and cyclin B1 were dramatically suppressed in Raji-KD cells,resulting in G₂/M arrest.In contrast to Raji cells,cell proliferation was significantly reduced in Raji-KD cells,and recovered via reintroduction of the c-Myc gene.In tumorigenesis assays,the loss of c-Myc expression significantly suppressed Raji cell-derived lymphoblastic tumor formation.Although cMyc also promotes Raji cell apoptosis via caspase 3-associated pathway,CDK1/cyclin B1-dependent-G₂/M cell cycle progression is still the main driving force of c-Myc-controlled tumorigenesis.Our results suggested that c-Myc could regulate G₂/M arrest as well as the expression of cyclin B1 and CDK1 by modulating HAT-mediated Ac H₄ in Raji cells and B-ALL tumorigenesis.Objective :To establish a recombinant retroviral vector expressing short interference RNA?si RNA?and study the function of c-Myc with RNA interference technology.Methods Using recombinant DNA techniques,complementary 82 bp oligonucleotides designed with hairpin loop for si RNA were annealed,and then inserted into the retrovirus expression vector p SINsi-h U6.Positive vectors were analyzed through digestion with Bam H I and Cla I.Recombinant retroviral vector p SINsi-h U6-c-Myc was generated by a insertion of 82 bp oligonucleotides designed with hairpin loop for si RNA into retroviral vector p SINsi-h U6.Raji cells were infected with the viral supernatant from the highest productive 293 T cells.Methods: Real time PCR was used for the detection of c-Myc m RNA expressionand,cMyc protein was detected with Western Blotting.Real-time PCR and RT-PCR assay the gene expression.using a FACS calibar instrument assay the Cell-cycle and apoptosis.Six-week-old male SCID mice were maintained in SPF environment.To establish the xenograft tumor formation model.Chromatin immunoprecipitation?Ch IP?to study the relationship between Ac H₄ and cycle gene.Results: The restriction enzyme analysis with Bam H I and Cla I showed that the recombinant retroviral vector had been constructed correctly.The plasmid was identified by PCR,and then DNA sequencing demonstrated the correct orientation and sequence.The titer assayed on 293 T cells was up to 2.1×105 CFU/ml.Using the RTPCR,Western Blotting,the c-Myc m RNA,protein and were down-regulated in TGP-49 cells after p SINsi-h U6-Fut8 infection.Loss of the MYC gene significantly reduces the growth rate of Raji cells.Loss of c-Myc decreased the expressions of cellcycle-associated genes and induced G₂/M arrest.c-Myc induces cell-cycle-associated gene expressions by transcriptionally regulating HAT.Knockdown of c-Myc suppressed apoptosis in Raji-KD cells by regulating caspase 3 expression.Tumorigenic effects of Raji cells were blocked by the knockdown of c-Myc in a SCID mouse model.Conclusion: The Raji-c-Myc-KD derivative cell lines stably transfected with the pSINsi-h U6-c-Myc plasmid-expressing siRNA that targeted c-Myc are established successfully.The study show that c-Myc could regulate G₂/M arrest as well as the expression of cyclin B1 and CDK1 by modulating HAT-mediated Ac H₄ in Raji cells and B-ALL tumorigenesis.