Study On The Anti-tumor Function And Mechanisms Of CHD5 In Chronic Myeloid Leukemia

Chronic myeloid leukemia(CML)is a common malignant myeloproliferative disorder of hematopoietic stem cells that accounts for 15%-20%of all cases of leukemia in adults.But so far,the reason and mechanism of this disease still are unclear.However,Philadelphia(Ph)chromosomes was discovered in 90-95%of patients.The Ph chromosome was the product of a t(9;22)(q34;q11)reciprocal translocation between chromosomes.This translocation generates the BCR-ABL fusion oncogene,which encodes the pathogenic protein with tyrosine kinase activity.The tyrosine kinase inhibitor represented by imatinib mesylate(IM)is designed targeted at this protein,and is the current standard treatment plan for CML.Despite the success of TKIs,the search for new therapeutic options in this disease entity is still important due to the emergence of primary or secondary resistance to treatment in patients and the difficulty of eradicating CML stem cells as the main "culprit" of the disease.Therefore,more researches should aimed at improving the the pathogenesis and molecular mechanism of chronic myeloid leukemia.Paving the way for better diagnostics and medications.The 1p36 is deleted in a broad range of human cancers,which suggestes that one or more tumor suppressor genes located in 1p36 are lost or inactivated.Chromodomain helicase DNA binding protein 5(CHD5)was previously proposed to function as a potent tumor suppressor locate on 1p36.The CHD5 protein belongs to chromatin remodeling enzyme,and its different domains can be combined with different positions of DNA or histone to change the structure of chromatin,reshape chromatin and promote gene transcription.Moreover,it controls cell proliferation,apoptosis and cellular senescence,all of which are dependent on p19Arf/p53 and p16/Rb pathway,acting as a master regulator of a tumor-suppressive network.At present,CHD5 missing has been found in various kinds of tumors like neuroblastoma,glioma,breast cancer and lung cancer and scientists also find that the low expression is related to tumor occurrence,development and prognosis.Thus,it is very significant to investigate the role and possible molecular mechanisms of CHD5 in different types of human tumors.Our previous study determined the expression of CHD5 in clinical samples and cell lines of acute myeloid leukemia(AML),acute lymphocytic leukemia(ALL),and chronic myeloid leukemia.We found that expression of CHD5 gene was down-regulated in leukemia cell lines and samples and repression of CHD5 gene expression in human leukemia is mediated in part by DNA methylation of its promote,suggesting that CHD5 plays a tumor suppressive role in leukemia.Therefore,our present study chose chronic myeloid leukemia as the entry point to explore the expression of CHD5 in chronic myeloid leukemia cell lines,the biological function and its possible molecular mechanisms.In this study,we used real-time quantitative PCR(RT-qPCR)and Western blotting to detect the expression of CHD5 in normal bone marrow mononuclear cells and chronic myeloid leukemia cell lines(KU812,K562 and KBM5).Compared with the normal bone marrow mononuclear cells,KU812,K562 and KBM5 cells had markedly lower CHD5 expression.Expression of CHD5 in CML samples was also lower than that of normal mononuclear cells.These results showed CHD5 expression is down-regulated in chronic myeloid leukemia.We applied CRISPR/dCas9-SAM technology to activate the endogenous CHD5 gene expression in CML cell lines(K562,KBM5).Subsequently,we observed the effect of CHD5 restoration on proliferation,cell cycle distribution,cell senescence,and cell differentiation by CFSE,soft agar plate clone formation,flow cytometry experiments and Senescence β-Galactosidase Staining.The TUNEL was used to detect the change of the apoptosis rate of chronic myeloid leukemia cells after CHD5 overexpression.The effect of CHD5 on autophagy was analyzed by transmission electron microscopy and Western blotting.Finally,we detected the anti-tumor effect of CHD5 restoration in tumor xenograft.The results showed that CHD5 restoration significantly inhibited cell proliferation and tumor growth,and induced G2/M phase arrest and apoptosis of chronic myeloid leukemia cell line,compared with the negative control group,while the cell senescence rate,autophagy and differentiation were not significantly effect.In order to elucidate clarify CHD5 molecular mechanism in regulating the progression of chronic myeloid leukemia,we used Western blotting to further analyze the expression of G2/M checkpoint(Cyclin B-cdc2)and cycle-related proteins CDK7,p53 and p21.The results indicated that CHD5 may inhibit the proliferation and induce apoptosis by up-regulating the expression of cdc2 phosphorylation and p21 in chronic myeloid leukemia.In summary,our study demonstrated that CHD5 was down-regulated in chronic myeloid leukemia compared with normal bone marrow mononuclear cells.CHD5 restoration can suppress cell proliferation and induce apoptosis of CML cells by up-regulatiing cdc2 phosphorylation and p21.Therefore,CHD5 is a tumor suppressor gene in chronic myeloid leukemia,so it could serve as a new therapeutic target and potentinal prognosis for CML patients.