Effects Of Knockdown MBD2 On The Cell’s Biology Behavior In K562 Cells

Objective: Chronic myeloid leukemia is characterized by the formation of BCR / ABL fusion genes which can encode fusion proteins with tyrosine kinase activity,causing abnormal clonal proliferation of hematopoietic stem cells.With the presence of imatinib,a targeted inhibitor of tyrosine kinase,90% of CML patients achieved long-term disease-free survival.However,with the application of imatinib,there have been many problems,the most prominent of which is imatinib resistance,in the study of resistance to imatinib,we found that about 30% of imatinib-resistance result from the kinase domain gene mutation,but many patients who do not undergo mutations in the kinase domain also develop resistance,which requires a mechanism to study drug resistance from other perspectives.A recent study of methylation of CpG gene in CML patients revealed that hypermethylation of some genes(such as CDKN2 B,OSCP1,PGRA,PRGB,TFAP2 E,etc.)was associated with poor prognosis and drug resistance in CML patients.And epigenetics mainly from the perspective of genome modification to study the mechanism of gene expression regulation,which caused epigenetic changes do not involve changes in gene sequences.DNA methylation is one of the important mechanisms of epigenetic modification,which is mainly concentrated in the promoter region of the CPG island methylation,and this apparent modification requires methylation of the protein family to mediate,MBD2 as an important member of the MBD family play a huge role.Therefore,it is important to study the effect of knockdown MBD2 on chronic myeloid leukemia cell lines.Methods: We use the CRISPR / Cas9 technique to design the gene editing tool,construct the tool plasmid of the silent MBD2 gene.The plasmid was transformed into the K562 cell line(human chronic myeloid leukemia cell line)by electroporation,and the plasmid expressed as a restriction endonuclease to cut target genes,in order to achieve the purpose of gene silence.Next,wild type and mutant cell lines were selected by flow monoclonal sorting and sequencing.The silencing effect was tested by RT-PCR and Western-Blot.Then,cell proliferation was detected by flow cytometry,cell cycle,CCK8 method and CFDA SE staining.Cell cloning ability was detected by colony formation assay.The tumorigenesis ability of nude mice was measured.Result: The MBD2 plasmid with silencing effect was successfully constructed by CRISPR / Cas9 technique.The stable passage of K562 cell line with silenced MBD2 gene was obtained by electric transfer sorting and sequencing.Through the study of its biological behavior,we found that the apoptosis of K562 cells did not change significantly after the MBD2 gene was silenced,but the cells showed obvious cycle arrest and most of them blocked at G0 / G1 phase.The abilities that the clonal formation ability and tumorigenic ability in nude mice in vivo are significantly inhibited.Conclusion: CRISPR / Cas9 technology can construct a unified gene silencing model of cell background.MBD2 gene in chronic myeloid leukemia cells in silence,reducing its characters of tumor stem cells.The mechanism may be due to DNA methylation reader MBD2 protein deletion,resulting in abnormal expression of DNA methylation spectrum.