The Effect Of M~6A Methyltransferase KIAA1429 On The Malignant Biological Phenotype Of Chronic Myeloid Leukemia And Its Mechanism

Chronic myeloid leukemia(CML)is a malignant clonal myeloproliferative disease by main abnormal proliferation of mature myeloid cells,characterized by t(9;22)translocation to form Bcr/Abl1 fusion gene.The natural history of CML is divided into chronic phase,accelerated phase and blast phase.In recent years,the widespread use of tyrosine kinase inhibitors(TKIs)such as Imatinib has greatly improved the prognosis of CML patients.However,some patients are resistant to TKIs,so TKIs cannot completely prevent the progression of CML.In addition,once CML progresses to blast phase,TKIs treatment is ineffective and the mortality rate is extremely high.At present,the mechanisms of TKIs resistance and blast crisis in CML patients have not been fully elucidated.Therefore,exploring the molecular mechanism of CML resistance and blast crisis and finding new therapeutic targets have become the focus of current CML research.In recent years,some new breakthroughs in epigenetics in tumor molecular targeted therapy have also provided new ideas for CML treatment.N~6-methyladenine(m~6A)modification is the most abundant in eukaryotes.m RNA modification is a dynamic and reversible modification process jointly regulated by methyltransferase complexes,demethylases and recognition proteins,regulating biological processes such as growth,reproduction,and meiosis.Studies have shown that m~6A modification and abnormalities of regulated genes are closely related to malignant biological phenotypes such as proliferation,apoptosis and drug resistance of hematological tumors.KIAA1429 is an m~6A methyltransferase that catalyzes m~6A methylation and other biological functions,and plays an important role in the occurrence and development of various tumors,but its research in CML has not been reported.Therefore,this study mainly analyzed the expression characteristics of KIAA1429 in CML from the four levels of clinical case,molecule,cell and animal,to clarify the molecular mechanism of KIAA1429/YTHDF1 m~6A axis regulating RAB27B expression and promoting the malignant phenotype of CML cells,and to find the anti-tumor effect of inhibiting KIAA1429 drug.The relevant research results will provide new ideas and targets for the treatment of CML.Chapter 1:Expression of KIAA1429 in CML and its effect on cell biological behaviorObjective:To investigate the expression of KIAA1429 in CML and its effect on the biological behavior of CML cells.Methods:Colorimetric method was used to detect the level of total RNA m~6A in peripheral blood mononuclear cells(PBMCs)of CML patients and CML cell lines,and real-time quantitative PCR(RT-q PCR),Western blot and immunohistochemistry were used to detect KIAA1429 m RNA in PBMCs of CML patients and protein expression.The Imatinib-sensitive cell line K562、KCL22 and the drug-resistant cell line K562/G01 were selected,and the CML cell model overexpressing or silencing KIAA1429 was constructed by lentiviral transfection.The effects of KIAA1429intervention on CML cell proliferation,apoptosis,migration and cell morphology were detected by Rig-Gill staining and Transwell assay.The effect of overexpression or silencing of KIAA1429 on Imatinib resistance of CML cells was detected by IC50.Results:The level of total m~6A in PBMCs of CML blast stage patients was significantly higher than that of CML initial stage patients,and the total m~6A level of K562/G01 cells was significantly higher than that of K562 cells.The expression of KIAA1429 was significantly up-regulated in CML blastic stage patients compared with CML initial stage patients,and the expression level of KIAA1429 in K562/G01cells was significantly higher than that in K562 cells.Overexpression of KIAA1429can up-regulate the level of total RNA m~6A in CML cells,promote CML cell proliferation,migration and Imatinib resistance,and inhibit cell apoptosis and differentiation.Silencing KIAA1429 can down-regulate the level of total RNA m~6A in CML cells,inhibit the proliferation,migration and Imatinib resistance of CML cells,and promote cell apoptosis and differentiation.Conclusion:The total m~6A level and the expression level of KIAA1429 were significantly up-regulated in CML blast stage patients.KIAA1429 can promote the proliferation,migration and Imatinib resistance of CML cells.It was shown that KIAA1429 is an oncogene of CML.Chapter 2: The KIAA1429/YTHDF1 m~6 A axis promotes the malignant biological behavior of CML cells by mediating RAB27 B m RNA stabilityObjective: To investigate the mechanism of KIAA1429/YTHDF1 m~6 A axis promoting the malignant biological behavior of CML cells by regulating RAB27 B through in vitro and in vivo experiments.Methods: K562 cells silenced by KIAA1429 and control cells were collected for RNA-seq identification,and combined with Me RIP-seq to screen target genes with m6 A modification downstream of KIAA1429.The expression level of KIAA1429 target gene RAB27 B m RNA in CML patients was detected by q PCR,and the correlation was analyzed.RT-q PCR and Western blot were used to detect the effect of KIAA1429 intervention on the m RNA and protein expression of RAB27 B.The binding of KIAA1429 to its target gene RAB27 B was verified by RIP-q PCR.A silent RAB27 B lentivirus was constructed and transfected into K562 cells to detect changes in cell proliferation,apoptosis,migration,drug resistance and drug efflux.Silencing RAB27 B was used to observe whether silencing of RAB27 B could restore the promotion effect of KIAA1429 on the biological behavior of CML cells.The YTHDF1-silencing lentivirus was constructed and transfected into K562 cells.RT-q PCR and Western blot were used to detect the changes of RAB27 B m RNA and protein expression after silencing YTHDF1.The binding of YTHDF1 protein to RAB27 B was verified by RIP-q PCR.The tumor nude mouse model was constructed by silencing KIAA1429,RAB27 B and YTHDF1.The changes of tumor volume and body weight of nude mice were detected,and the effect of silencing target gene expression on the tumorigenic ability of CML cells in nude mice was evaluated.Results: The expression of RAB27 B was up-regulated in CML blastic stage patients compared with CML initial stage patients,and was positively correlated with the expression of KIAA1429.Silencing KIAA1429 can reduce the stability of RAB27 B m RNA and down-regulate its expression.RIP-q PCR confirmed thatKIAA1429 protein can directly bind to RAB27 B m RNA.Silencing RAB27 B can inhibit the proliferation and migration of CML cells,lead to S-phase arrest of cells,inhibit the efflux of Imatinib,reduce the IC50 of Imatinib resistance in cells,and partially reverse the effect of KIAA1429 overexpression on the malignant expression of CML cells.type of promotion.The expression levels of RAB27 B and YTHDF1 were positively correlated,and silencing YTHDF1 could reduce the stability of RAB27 B m RNA,thereby reducing the expression levels of RAB27 B m RNA and protein.RIP-q PCR confirmed that YTHDF1 could directly bind RAB27 B m RNA.K562 cells silenced KIAA1429,RAB27 B,and YTHDF1 were inoculated into nude mice.Compared with the blank control group,the tumorigenic rate and tumor volume of mice in the silenced KIAA1429,RAB27 B and YTHDF1 silenced groups were significantly decreased,which inhibited the formation of K562 cells in nude mice.tumor capacity.Conclusion: The KIAA1429/YTHDF1 m6 A axis can up-regulate the expression of RAB27 B by mediating the stability of RAB27 B m RNA,and promote the occurrence and development of CML.Chapter 3: Rucaparib can inhibit the expression of KIAA1429,then inhibit the proliferation of CML cells and promote cell apoptosisObjective: To investigate the effect of Rucaparib on the expression of KIAA1429 and the occurrence and development of CML through in vitro and in vivo experiments.Methods: Bioinformatics analysis was used to predict antitumor drugs and small molecular compounds with similar molecular mechanism of KIAA1492.After CML cells were treated with concentration gradient Rucaparib,the expression levels of KIAA1429 m RNA and protein were detected by RT-q PCR and Western blot,and the proliferation,apoptosis and drug resistance were detected by CCK-8,flow cytometry and IC50.CML cells were co-treated with Rucaparib and Imatinib,and their proliferation,apoptosis and drug resistance were detected by CCK-8,flow cytometry and IC50.The drug experiment of Rucaparib was carried out on nude mice,and the therapeutic effect of Rucaparib on CML in vivo was evaluated by detecting the changes of tumor volume and body weight of nude mice.Results: Bioinformatics prediction showed that the control group was more sensitive to the antitumor drugs Ponatinib,Rucaparib,Axitinib and ATRA than the KIAA1429 silenced group.Rucaparib can inhibit CML cell proliferation and promote CML cell apoptosis in a dose-dependent manner.After treatment with Rucaparib,the expression levels of KIAA1429 protein and m RNA in CML cells were down-regulated,and the degree of down-regulation was positively correlated with the concentration of Rucaparib.The combined use of Rucaparib and Imatinib can reduce the IC50 of Imatinib resistance of CML cells,and the apoptosis rate of CML cell lines is significantly higher than that of the two alone.Compared with the control group,the tumor volume growth rate of nude mice in the Rucaparib administration group was significantly decreased,and Rucaparib could inhibit the tumorigenic effect of CML cells in vivo.Conclusion: Rucaparib can inhibit the expression of KIAA1429,inhibit the proliferation of CML cells,and promote cell apoptosis,and the combination of Rucaparib and Imatinib can improve the sensitivity of CML cells to Imatinib.Rucaparib inhibits the tumorigenic ability of CML cells in vivo.