The Role Of CD96 In The Occurrence And Development Of Acute Myeloid Leukemia

Background:Acute myeloid leukemia(AML)is one of the most common malignant acute leukemia in adults.Currently traditional treatments of AML are the combination of chemotherapy,hematopoietic stem cells transplantation and so on.However,the traditional methods not only have obvious side effects,but also low patient's long-term survival rate or complete remission rate.It means that the development of new treatment methods is imminent.Over the past 20 years,a large number of reports have reported that Leukemia stem cells(LSCs)are regarded as the root of leukemia origin and leukemia recurrence.LSCs are derived from normal hematopoietic stem cells(HSCs),so that they share many similar phenotypes and biological characteristics with HSCs.The majority of LSCs stay in quiescent phase and maintain self-renewal in vivo,that is an important reason that they can acquire drug resistance.The xenotransplantation of primary CD34~+CD38~-cells isolated from AML patient samples demonstrated strong engraftment in mice,suggesting that LSCs and normal HSCs have similar immunophenotypes.New ideas for treatment of AML by using the LSCs specific cell surface molecules as a target of fixed-point cleaning,if screening of specific cell surface molecules of LSCs is feasible,that reducing the negative impact on the hematopoietic stem cells.In our previous study,we have found that the high expression of surface molecules were selected from a large number of clinical samples,which are low or no expression on hematopoietic stem cells in normal bone marrow,and to explore the mechanism of specific surface molecules in the occurrence and development of AML.Objective:To study the role of molecule CD96 in the development of acute myeloid leukemia.Methods:In order to find the relationship between CD96 expression and survival rate of AML patients,we used three databases:TCGA,GSE6891,and GSE10358.We further identified the expression level of CD96 in 8 different subtypes of AML.The shRNAs(shScramble,shCD96)were designed,constructed a lentiviral vector and transfected into the packaging cell 293T to produce a lentivirus;lentivirus was transfected into AML cells such as U937 and THP-1 to knockdown CD96.Then,GFP-positive cells mean successfully transfected were sorted by using a flow cytometric sorter.The number of cells in the GFP~+shScramble group and the GFP~+shCD96 group were respectively calculated by cell counting at different time points,and the cell growth curve was drawn;the ability of cell clone formation was detected by cell colony formation assay;the differentiation ability of the GFP~+shScramble group and the GFP~+shCD96 group were detected by an immunostaining method and Giemsa staining was used to detect the morphological differences between GFP~+shScramble group and GFP~+shCD96 cells.The GFP~+shScramble group and the GFP~+shCD96 group cells were sorted by flow cytometric sorter,then transplanted GFP~+cells into mice.After 15 days,the engraftment ability of the cells in the peripheral blood,bone marrow,liver,and spleen of the mice was detected.Results:Through three databases analyzing,CD96 expression level was negatively correlated with AML patient's survival:CD96 was lowly expressed,and the patient's survival rate was higher.Furthermore,CD96 was expressed in all subtypes of AML,especially in AML-M4 and AML-M5 subtypes.Compared with GFP~+shScramble group cells,when CD96was knockdown in AML cell lines including THP-1 and U937,it obviously inhibited cell proliferation.Meanwhile the ability of colony formation decreased,but the expression of CD11b and CD19 antigens increased.The results of mouse xenograft experiments showed that the volume of liver and spleen of GFP~+shScramble group was significantly greater than that of GFP~+shCD96 group,and the percentage of GFP~+cells in liver was significantly decreased in GFP~+shCD96 group compared with control group.Conclusion:AML cells highly expressed CD96,which made cells maintain self-renew,inhibited AML cells apoptosis and blocked AML cells differentiated into mature cells,so that facilitates AML development.The results of mouse xenograft experiments indicated it is possible that CD96 is involved in the regulation of AML cells engraftment within mice.This study is to further clarify the molecular mechanism of AML development,and add new contents to find the effective target treatment in AML.