The Role Of C-Myc Transcription Inhibitory Activity In The Occurrence And Development Of Leukemia

C-Myc is a classical proto-oncoprotein,which is mainly involved in regulating cell proliferation,differentiation,apoptosis,survival and adhesion,cell size and other important cellular biological functions.A large number of studies have shown that c-Myc is the common target gene of most leukemia fusion genes,which is highly expressed in the leukemia cells of most patients and essential for the occurrence and development of leukemia.C-Myc protein is involved in regulating the self-renewal,proliferation and differentiation of hematopoietic stem/progenitor cells mainly through transcriptional activation and transcriptional inhibition.The transcriptional activation of c-Myc mainly promotes cell cycle,stimulates cell growth and metabolism,and plays a key role in tissue repair and reconstruction and the occurrence and development of tumors.Transcriptional inhibition of c-Myc mainly regulates downstream target genes by binding Miz1,but little is known about its biological significance.It was found that the 394th amino acid mutation of c-Myc protein,namely c-Myc ^V394D?the valine mutation is aspartic acid?,was not able to bind to Miz1,so its transcriptional inhibitory activity was inactivated,but its transcriptional activation function was still normal.Therefore,c-Myc ^V394Dmutation provides an effective experimental tool for us to specifically study the biological effects of c-Myc transcription inhibition function in the development of leukemia.In this study,we used retroviruses to infect hematopoietic stem/progenitor cells andconstructedcelllinesoverexpressedwithMSCV-c-Myc-GFPand MSCV-c-Myc^V394D-GFP.Through a series of experiments in vivo and in vitro,we studied the role of c-Myc transcriptional inhibitory activity in the development of leukemia.First,we established animal models of c-Myc and c-Myc ^V394D394D overexpression by tail vein transplantation,observed their survival,analyzed the changes of peripheral blood cells and cell infiltration,and studied the role of c-Myc and c-Myc ^V394D394D overexpression in the development and progression of leukemia.In addition,in vitro culture conditions,we explored the effect of c-Myc and c-Myc ^V394D394D overexpression on the proliferation capacity of hematopoietic stem/progenitor cells through cell cloning formation experiments.The effects of c-Myc and c-Myc ^V394D394D overexpression on the cycle,apoptosis and differentiation of hematopoietic stem/progenitor cells were analyzed by flow cytometry.At the same time,the transcriptional regulation effect of c-Myc and c-Myc ^V394D394D on the downstream target genes was detected by qRT-PCR technology,and the molecular regulatory mechanism of c-Myc transcriptional inhibitory activity in the development of leukemia was explored.The results of in vivo experiments showed that c-Myc overexpression could induce the development of acute myelogenous leukemia,while c-Myc ^V394D394D overexpression could not induce leukemia in mice.The mice with c-Myc overexpression developed disease at the second month and all died at the fourth month.The mice with c-Myc ^V394D394D overexpression and the control group were able to survive normally.At the same time,compared with the control group,the proportion of GFP⁺cells in mice with c-Myc overexpression was significantly increased,the number of white blood cells and platelets was decreased,the number of granulocytes was abnormally increased,the liver and spleen were enlarged,and leukemia cells were infiltrated,which proved that c-Myc overexpression promoted the occurrence and development of leukemia.The results of in vitro experiments showed that the overexpression of c-Myc could enhance the proliferation capacity of hematopoietic stem/progenitor cells and inhibit the ability to differentiate into granulocytes,thus enabling the continuous proliferation of cells.However,c-Myc ^V394D394D could not maintain the continuous proliferation of hematopoietic stem/progenitor cells.It was found that the clonal formation ability of c-Myc and c-Myc ^V394D394D overexpressed hematopoietic stem/progenitor cells was significantly enhanced.From the second generation of clone formation,the clonal formation ability of the hematopoietic stem/progenitor cells with c-Myc overexpression was significantly higher than that of the c-Myc ^V394D394D overexpression group and the control group,until the fourth generation of clone formation,the control group and the hematopoietic stem/progenitor cells with c-Myc ^V394D394D overexpression almost lost their clonal formation ability.Flow analysis showed that overexpression of c-Myc and c-Myc ^V394D394D had no significant effect on apoptosis.However,in the study of cell cycle progression and cell differentiation,it was found that c-Myc overexpression could induce cells to enter S/G2 phase from G1 phase,promote cell cycle progression,and enable cells to maintain the expression of c-Kit antigen,thus hinding the pathway of cell differentiation into granulocytes;While,overexpression of c-Myc^V394D394D could not promote cell cycle progression,and the proliferation and differentiation functions were normal.In addition,we found that c-Myc overexpression inhibited the expression of C/EBP?,while c-Myc ^V394D394D overexpression could upregulate the expression of C/EBP?,and both could inhibit the expression of p15 and p21.Therefore,the transcriptional inhibitory activity of c-Myc mainly promotes the process of cell cycle,stimulates the continuous proliferation of hematopoietic stem/progenitor cells and the dysfunction of granulocyte differentiation,thus inducing the occurrence and development of acute myelogenous leukemia.