| Acute myeloid leukemia(AML)is a disease caused by malignant clonal proliferation of myeloid hematopoietic stem cells.Its two pathological features are blocked differentiation and uncontrolled malignant proliferation.The disease can occur in people of all ages.The disease deteriorates rapidly and the clinical prognosis is very poor,and the mortality rate is very high.In clinic,arsenic trioxide(As2O3)and all trans retinoic acid(ATRA)are mainly used to treat the M3 classification of the disease,namely acute promyelocytic leukemia(APL),and achieved good therapeutic effect.However,ATRA is not effective in the treatment of other types of AML(non-APL),and it has serious drug resistance,retinoic acid syndrome and recurrence rate.In order to solve the above problems,our team carried out structural modification on ATRA and synthesized a series of all trans retinoic acid derivatives.After preliminary pharmacodynamic screening,we found that 4-amino-2-trifluoromethyl-phenyl retinate(ATPR)has a good therapeutic effect,not only for APL(NB4)but also for non-APL(THP-1).However,the specific mechanism of ATPR in the treatment of AML is still unclear and needs further studyIn order to further study the molecular mechanism of ATPR in the treatment of AML,we used whole transcriptome sequencing to screen out differentially expressed mRNA and long noncodingRNA(lncRNA).lncRNA is a kind ofRNA with a length of more than 200 bp,which does not have the ability to encode proteins.The latest research results show that lncRNA plays an important role in the process of cell proliferation,differentiation,and apoptosis,and may become a molecular marker of tumor,and participate in various pathological processes such as tumorigenesis and development.Therefore,ATPR may play a role in the treatment of AML by regulating the expression of specific lncRNA.This paper mainly includes the following two parts.Aims:In this study,a new type of lncRNA NR-104098 was screened through the whole transcriptome gene chip technology,aiming to explore the role and mechanism of NR-104098 in the process of ATPR inhibiting AML cell proliferation,inducing cycle arrest and differentiation,and providing new treatments for AML Ideas and experimental basis.Methods:1 The whole transcriptome analysis of ATPR induced acute myeloid leukemia NB4 cells1.1 Whole transcriptome sequencing:Agilent single-channel expression profiling chip technology was used to screen NB4 cells in the Control group and ATPR group.The quality of the sequencing results was detected,and the differential expression of lncRNA was analyzed by thermography and volcano map.The function and signal pathway of differentially expressed lncRNAs were analyzed by GO and KEGG enrichment analysis methods.1.2 qRT-PCR was used to identify the sequencing results:After the totalRNA of control group and ATPR group was extracted,the five lncRNAs with the most obvious up-regulation and the five most obvious down-regulation were detected by qRT-PCR for quantitative analysis.2 The role of NR-104098 in ATPR inhibiting the proliferation of acute myeloid leukemia cells,inducing cycle arrest and differentiation2.1 The low expression of NR-104098 in AML cells:qRT-PCR was used to analyze the expression changes of NR-104098 in different AML cells(KG-1,NB4,U937,HL-60,THP-1).2.2 NR-104098 inhibits AML cell proliferation,induces cycle arrest and differentiation:Transfection with over-expression lentivirus up-regulates the expression level of NR-104098 in AML cells,qRT-PCR detects the expression level of NR-104098;CCK8 test detects the proliferation ability of AML cells;Immunofluorescence test to detect the expression of proliferation-related protein ki67;flow cytometry to detect the distribution of cell cycle;flow cytometry to detect the proportion of positive cell surface differentiation antigens CD11b and CD14;Western blotting to detect related P-Rb,CDK4,Cyclin A2,Cyclin D3,PCNA,CD11b and CD14 protein expression.2.3 NR-104098 inhibits the proliferation,induces cycle arrest and differentiation of transplanted tumors in NCG mice:In order to explore the effect of NR-104098 in vivo,a NB4 cell line stably overexpressing NR-104098 was constructed in vitro,and the tumors were inoculated subcutaneously in NCG mice.Tissue,measure tumor weight,Western blotting to detect the expression of P-Rb,CDK4,Cyclin A2,Cyclin D3,CD11b,and CD14 protein.2.4 ATPR inhibits AML cell proliferation,induces cycle arrest and differentiation through NR-104098:Different gradient concentrations(10-5,10-6,10-7,10-8,10-9M;72h)of ATPR,different time points(0,24,48,72 h;10-6M)ATPR was applied to AML cells in vitro,and qRT-PCR was used to analyze the expression changes of NR-104098in AML cells;NR-104098 shRNA was transfected in AML cells,After silencing the expression of NR-104098,give ATPR(10-6M)stimulation for 72 h,qRT-PCR was used to analyze the expression changes of NR-104098 in AML cells;CCK8 test cell proliferation ability;immunofluorescence test to detect the expression of proliferation-related protein ki67;flow cytometry Detection of cell cycle distribution;flow cytometry detection of cell surface differentiation antigen CD11b and CD14positive cell ratio;Western blotting detection of related P-Rb,CDK4,Cyclin A2,Cyclin D3,PCNA,CD11b and CD14 protein expression.2.5 ATPR inhibits the proliferation,induces cycle arrest and differentiation of transplanted tumors in NCG mice through NR-104098:In order to explore the therapeutic effect of ATPR on AML in vivo and the effect of NR-104098 shRNA on the treatment process,NCG mice are divided into NC shRNA,NC shRNA+ATPR and NR-104098 shRNA+ATPR groups,the expression of P-Rb,CDK4,Cyclin A2,Cyclin D3,CD11b and CD14 were detected by Western blotting.3 The mechanism of NR-104098 on the ATPR inhibiting the proliferation of acute myeloid leukemia cells,inducing cycle arrest and differentiation3.1 NR-104098 can regulate the expression of EZH2:Overexpression lentivirus was used to up-regulate the expression of NR-104098 in AML cells;Western blotting and qRT-PCR were used to detect the protein and mRNA expression of EZH2;Luciferase reporter gene assay was used to detect the promoter activity of EZH2;Western blotting and immunohistochemistry were used to detect the expression of EZH2 protein in transplanted tumors of NCG mice.The shRNA of NR-104098 was transfected into AML cells,and the expression of NR-104098 was silenced,and then treated with ATPR(10-6M)for 72 h;Western blotting and qRT-PCR were used to detect the protein and mRNA expression of EZH2;Luciferase reporter gene assay was used to detect the promoter activity of EZH2;Western blotting and immunohistochemistry were used to detect the expression of EZH2 protein in transplanted tumors of NCG mice.3.2 NR-104098 can regulate the expression of EZH2 by binding with E2F1:Overexpression lentivirus was used to up-regulate the expression of NR-104098 in AML cells.CHIP experiment was used to detect the binding ability of E2F1 and EZH2promoter regions;Western blotting and qRT-PCR were used to detect the protein and mRNA expression of E2F1.Small interferingRNA was used to silence the expression level of E2F1 in AML cells,and the protein and mRNA expression levels of EZH2 were detected by Western blotting and qRT-PCR.RIP andRNA-pull down experiments detect the combination of NR-104098 and E2F1.Results:1.Compared with control group,8662 differentially expressed lncRNAs and 9093differentially expressed mRNAs were identified in ATPR group.2.qRT-PCR results were consistent with sequencing results,and the sequencing results were reliable.3.ATPR could activate the expression of NR-104098 and promote its transfer from cytoplasm to nucleus,and the expression of NR-104098 was low in AML cells.4.Overexpression of NR-104098 can inhibit AML cell proliferation,induce cycle arrest and differentiation:5.Silencing NR-104098 can reverse the therapeutic effect of ATPR on AML in vivo and in vitro.6.NR-104098 can regulate the expression of downstream target gene EZH2 by binding with transcription factor E2F1.Conclusion:1.The whole transcriptome sequencing results show that compared with the Control group,the mRNA and lncRNA expressions in the ATPR group are significantly different,and a new type of lncRNA NR-104098 was screened.2.ATPR activates the expression of NR-104098,induces its binding to the transcription factor E2F1,and regulates the expression of EZH2 protein to inhibit the proliferation of AML cells,induce cycle arrest and differentiation... |
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