The Roles Of Abnormal Epigenetic Of P53 And Oxidative Damage In The Resistance Of Chronic Myeloid Leukemia To Imatinib

Background:Chronic myeloid leukemia?CML?is a myeloid proliferative tumor originating from pluripotent stem cells.It is mainly characterized by the proliferation of myeloid cells in peripheral blood and bone marrow,chromosome translocation to form a characteristic Philadelphia chromosome and BCR-ABL fusion gene-positive.The BCR-ABL fusion gene is a characteristic alteration of the disease and encodes a BCR-ABL fusion protein with high tyrosine kinase activity.Imatinib?IM?is an effective tyrosine kinase inhibitor,which is currently used as a first-line treatment for CML.15%of patients in clinical practice are resistant to drug resistance.However,with the expansion of the scope of clinical application and the passage of time,drug resistance phenomenon is emerging,about15%of patients in clinical practice are resistant to drug resistance,which seriously affects the treatment effect of CML patients.Although there are many studies on the mechanism of resistance to IM,it has not yet been fully elucidated.Therefore,this study mainly focused on the relationship between p53 dysfunction and oxidative stress injury and Imatinib resistance in chronic myeloid leukemia.In this study,Imatinib-resistant cell lines were used in vitro and combined with in vivo experimental studies to elucidate the p53 gene abnormal epigenetic modification and the mechanism of oxidative stress injury in Imatinib-resistant chronic myeloid leukemia.Objective:The purpose of the study is to research the express of the related markers with the the p53 signaling pathway,the oxidative stress damage products and apoptosis levels of cells,the relationship between therapeutic response and differences in CML patients,so as to deeply understand the IM resistance mechanism of CML and explore the potential role of p53 gene and oxidative stress in CML,provide scientific experimental support for the new target treatment of CML.Methods:?1?In vitro,the chronic myelocytic leukemia cell line K562 was used as the research object,and the imatinib resistant cell line K562-G was gradually induced by the concentration gradient method.?2?After different concentrations of imatinib were applied to K562 and K562-G cell lines,the cell viability of the two cell lines was determined by CCK-8 assay,and the IC₅₀ values of the two cell lines against imatinib were measured to calculate the drug resistance multiple.?3?The concentrations and action time of imatinib,ROS inhibitor NAC and STAT5inhibitor SH-4-54 were determined by preliminary experiments,using the method of flow cytometry detection of two kinds of cell lines of apoptosis and the content of intracellular ROS.The content of?-H2AX,a marker of DNA double-strand breaks,in each experimental group was determined by immunofluorescence assay.?4?The mRNA level of STAT5A,STAT5B and internal reference GAPGH in CML cell line K562 and imatinib resistant cell line K562-G were detected by real-time fluorescence quantitative PCR?qRT-PCR?.?5?Western blot was used to analyze the protein levels of relevant signaling pathways,and to detect the expression of STAT5 protein and phosphorylated STAT5 protein in the two cell lines.?6?The peripheral blood of 63 patients with chronic myelogenous leukemia was collected,and the total RNA in the cells was extracted.The mRNA level of BCR-ABL?STAT5A?STAT5B?p53?p53BP1?GADD45A and RAD17 in the cells were detected by real-time fluorescence quantitative PCR.The mutation rate of BCR-ABL and p53 gene in CML patients and CML lines were detected by Sanger sequencing.?7?The change of p53 signaling pathway related markers,by real-time fluorescent quantitative PCR?qRT-PCR?method to detect the changes of p53,MDM2,SIRT1,Bax and Bcl-2 mRNA expression in each group.Western blot method was used to detect the expression of related proteins.Results:?1?Successfully established resistant imatinib cell line K562-G,and IC₅₀values of two cell lines were determined by CCK-8 assay.The extent of resistance to imatinib in K562-G cell line was 87 times as high as that of the parental cell line K562.?2?The apoptosis of K562 cells and K562-G cells showed significant difference?P<0.05?with different concentrations of imatinib,and the apoptosis of K562-G cells was significantly decreased.?3?Compared with K562 cell line,ROS levels in K562-G cells were significantly increased?P<0.05?,while reactive oxygen species?ROS?levels in K562-G+NAC and K562-G+SH-4-54 cells were significantly decreased?P<0.05?.At the same time,the level of?-H2AX in K562-G cells was significantly higher than that in K562 cells,while the level of?-H2AX in K562-G+NAC cells was significantly decreased.?4?There were significant differences in the expression levels of STAT5A and STAT5B mRNA in K562 and k562-G cells?P<0.05?,and the K562-G cells were significantly increased.?5?There were significant differences in STAT5 and phosphorylated STAT5 protein levels in K562 and k562-G cells?P<0.05?,and K562-G cells significantly increased.?6?The expression of STAT5A and STAT5B in CML patients was significantly higher than that in CML non resistant patients?P<0.05?,at the same time,the expression of STAT5A and STAT5B in CML patients with BCR-ABL mutation in drug-resistant and non-drug resistant groups were significantly higher than that in BCR-ABL non-mutated group?P<0.05?.?7?Compared with IM non-resistant group,mRNA expression levels of p53,p53BP1,GADD45A and RAD17 in CML patients with IM-resistant group decreased,especially p53 and p53BP1 mRNA expression levels decreased more significantly,the difference was statistically significant Significance?P<0.05??8?Among 63 CML patients,the mutation rate of BCR-ABL in non-drug-resistant group was 9.6%.Three kinds of point mutations were detected,including D276G,F317L and F359I.The mutation rate of BCR-ABL in drug-resistant group was 28.13%.Nine kinds of point mutations,including G250E,M244V,M387L,V299L,Y253H,E255K,E297K,F317L,G250E.No p53 gene mutation was detected in 63 CML patients.No mutations in the BCR-ABL and p53 genes were detected in CML cell lines.?9?Compared with K562 cells,the mRNA expressions of p53 and Bax in K562-G cells were significantly decreased,while the mRNA levels of Bcl-2,MDM2 and SIRT1 were significantly increased?P<0.05?,while the level of p53 protein in the cell K562 and K562-G are lower expression,almost undetectable.Compared with K562 cells,the level of Bax protein in K562-G cells decreased significantly,while the level of Bcl-2,MDM2 and SIRT1 protein increased significantly?P<0.05?.Conclusion:Due to the formation of BCR-ABL fusion gene in CML patients,BCR-ABL can act directly on STAT5 to induce high levels of ROS production,leading to chronic oxidative DNA damage and genomic instability of DNA,eventually leading to mutations of imatinib resistance-related genes when BCR-ABL act on STAT5,the increasing of ROS lead to DNA oxidative damage and increase the cell mutation,then cause CML imatinib resistant.P53 signaling pathway is an important DNA damage repair mechanism of the body,and p53 is also an important regulatory factor that mediates the induction of apoptosis induced by excessive ROS.P53 translational modification acetylation and ubiquitination can precisely regulate the activity of p53protein.The ubiquitination can regulate the degradation of p53 protein through MDM2,and can also inhibit the transcriptional activation of multiple lysine sites of p53 by deacetylation of p53.The study of epigenetic modification and oxidative stress damage of p53 gene provides new ideas for CML imatinib resistance,and provides scientific basis for exploring multi-target therapy for CML patients.