Study On The Mechanism Of Long-distance Expression Regulation Of C-myb In Human Leukemia Cells

As an important transcription factor during the differentiation and maturation of blood cells,c-Myb is related to the differentiation,proliferation and apoptosis of myeloid cells.Abnormal expression of c-Myb has been found in various malignant tumors such as leukemia,colorectal cancer and breast cancer.Although the mechanism of c-myb regulation in human and mouse leukemia cells has been studied to some extent,the mechanism of c-myb regulation in human leukemia cells is still unclear.C-myb participates in hematopoiesis and maturation and differentiation in human blood cells,mice and some mammals.The precise regulation of c-myb expression is unclear.The regulatory mechanisms and signaling pathways related to c-myb expression need further exploration.Overexpression of c-Myb is associated with human T-cell acute lymphoblastic leukemia.A significant increase in the number of hematopoietic stem cells was found in mice that reduced c-Myb expression.Although the proportion of myeloid cells such as erythrocytes and megakaryocytes increased in the blood of mice after c-myb was knocked out,the number still showed a significant downward trend when compared with absolute values.Based on the important role of c-myb in the differentiation and maturation of blood cells and the formation of leukemia,it is of great significance for the study of the regulatory mechanism of c-myb in human leukemia cells.In this experiment,human leukemia cells were used as the research object to investigate the transcription factors GATA1,GATA2,CTCF,TAL1,PU.1,C / in human red leukemia K562 cells,human acute myeloid leukemia U937 cells,and human promyelocytic leukemia HL60 cells.EBP?,Rad21,c-Jun regulate c-myb expression.First we induced U937 cells with 100 ?g / L TPA(12-oxo-tetradecanoyl phorbol-13-acetate)for 72 h,and K562 cells with 40 ?mol / L Hemin(chloroheme)for 24 h HL60 cells were induced with 2 ?mol / L ATRA(all trans retinoic acid)for 72 h.The expression changes of these transcription factors and c-myb were detected after induction,and it was found that the expression of GATA1,c-Jun,TAL1,C / EBP? and c-myb changed at the same time,which means that GATA1,c-Jun,TAL1,C / EBP? May be related to the expression of c-myb.The Ch IP-q PCR experiment was used to detect the significant changes in the enrichment of c-Jun at-34 K and-88 K before and after K562 cell differentiation,indicating that the transcription factor c-Jun has an effect on c-myb and-34 K and-88 K.c-myb upstream distal enhancer.We cloned the GATA1 gene and successfully constructed the p LVX-IRES-GATA1 lentiviral expression plasmid;at the same time,we designed the GATA1,TAL1,c-Jun,GATA2,CTCF,PU.1,and sh RNA knockdown plasmids for the Rad21 transcription factor and constructed it into p LKO.1 plasmids form p LKO.1-GATA1 sh RNA,p LKO.1-TAL1 sh RNA,p LKO.1-c-Jun sh RNA,p LKO.1-GATA2 sh RNA,p LKO.1-CTCF sh RNA,p LKO.1-Rad21 sh RNA,p LKO.1-PU.1 sh RNA plasmid.We used the dual luciferase reporter system to detect the GATA1 transcription factor at-34Ka(we divided the-34 K segment into three segments a,b,and c to form-34 Ka,-34 Kb,-34Kc)and-88 K region to The effect of c-myb promoter expression.293 T cells were packaged to produce lentiviruses with infection efficiency,and K562 and U937 cells were infected.The expressions of c-myb and transcription factors were detected by Western blot and RT-q PCR.The results showed that:(1)Western blot results showed that the expression of c-Jun,TAL1,C/EBP? in K562 cells was significantly increased after Hemin induction,the expression of GATA1 was significantly decreased,and the expression of c-Myb was significantly decreased;Ch IP-q PCR showed that K562 was induced after differentiation C-Jun binding in cells decreased in the c-myb promoter,-34 kb,and-88 kb regions;(2)Western blot results showed that after U937 cells were induced by TPA,the expression of c-Jun and TAL1 increased significantly,and the expression of GATA1 decreased significantly,c-Myb expression was significantly decreased;(3)Western blot results showed that C/EBP? expression was significantly increased in HL60 cells after ATRA induction,GATA1 expression was significantly decreased,and c-Myb expression was significantly decreased;(4)RT-q PCR and Western blot results showed success In K562 cells overexpressing GATA1,c-Myb expression also increased;RT-q PCR results showed that KATA cells that successfully knocked down GATA2,CTCF,PU.1,Rad21,TAL1,and c-Jun followed the expression of c-myb.decline.(5)RT-q PCR and Western blot results showed that c-Myb expression also increased and decreased in U937 cells that successfully overexpressed and knocked down GATA1;(6)Detection by dual luciferase reporter system showed that GATA1 can promote c-myb promoter expression.In summary,this experiment was designed to detect the changes in expression of c-myb and transcription factors GATA1,GATA2,CTCF,PU.1,Rad21,TAL1,and c-Jun by using inducers to induce the differentiation of leukemia cells K562,U937,and HL60.Transcription factor expression plasmids and knockdown plasmids.Lentiviral packaging infection technology was used to knock down or overexpress transcription factors in leukemia cells to further explore the regulatory effect of transcription factors on c-myb.The dual luciferase reporting system was used to further confirm GATA1 Regulating c-myb,and using the Ch IP-q PCR technology to explore the binding of c-Jun at the remote regulatory site upstream of c-myb.The above results show that GATA1 can promote the expression of c-myb in K562 and U937 cells,and GATA1 may promote the expression of c-myb in HL60 cells.After K562 cells were induced to differentiate,the expression of c-Jun,TAL1,C/EBP? increased significantly,and the expression of c-myb decreased.However,the phenomenon of c-myb decreased after knocking down these transcription factors in K562 cells.At present,it can only explain that The expressions of c-Jun,TAL1,and C/EBP? are related to c-myb expression in K562 cells.The specific mechanisms need to be further studied.GATA2,CTCF,PU.1,and Rad21 may promote c-myb expression in K562 cells.Based on the important role of c-myb gene in leukemia and other cancers and the regulation mechanism of c-myb expression by these transcription factors,this study can provide new ideas for leukemia research protocols and treatments.