The Expression And Function Of C-type Lectin-like Receptor 2 In Acute Myeloid Leukemia

Background and Objective:Acute myeloid leukemia(AML)is a common type of hematologic malignancy which characterized by abnormal expansion of myeloid blasts in the bone marrow and peripheral blood.The membrane protein C-type lectin-like receptor 2(CLEC-2)belongs to the C-type lectin domain superfamily,which is a class of tumor regulatory proteins.Related studies have confirmed that CLEC-2 and its ligands regulate tumor metastasis and invasion in solid tumors such as pancreatic cancer,lung cancer,etc.CLEC-2 is also involved in maintaining hematopoietic stem cell differentiation.However,the role of CLEC-2 in hematological tumors is unclear.Based on the analysis of RNA-seq database,it was found that the expression level of CLEC-2 in myeloid leukemia cells was lower than that in normal granulocytes.Therefore,we speculated that the loss of CLEC-2 might affect the normal differentiation of hematopoietic stem cells and their conversion to leukemia cells.In order to explore the expression changes,biological effects and related mechanism of CLEC-2 in AML,we detected the expression of CLEC-2 in AML patients through clinical specimens and verified the results of the high-throughput database.The expression of CLEC-2 in different subtypes and stages of AML were analyzed to explore the correlation between CLEC-2 expression and clinical phenotypes of AML patients.The expression level of CLEC-2 was regulated in vitro to verify the effect of CLEC-2 on various phenotypes of myeloid leukemia cell lines,and to explore the role of CLEC-2 in AML.Methods:(1)RNA-seq data for normal hematopoietic cell lines and blood tumor cell lines were obtained from the public databases Human Protein Atlas and CCLE.The difference of CLEC-2 expression between AML and normal hematopoietic cells were analyzed by network cross-analysis and multiple hypothesis testing.(2)Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the difference of CLEC-2 expression in peripheral blood mononuclear cells(PBMCs)of 58 newly diagnosed AML patients,contemporaneous healthy volunteers(normal control group)and newly diagnosed acute lymphoblastic leukemia patients(disease differential control group).Chi-square test was used to analyze the correlation between CLEC-2 expression level and clinical characteristics,molecular subtypes and status before and after treatment.Protein levels of CLEC-2 in plasma in newly diagnosed AML patients was detected and analyzed by ELISA.(3)Human myeloid leukemia cell line MV4-11,was selected to construct the CLEC-2overexpressed cell model(pCDH-CLEC-2)by lentiviral vector,with MV4-11 cells infected by lentiviral blank vector as negative control(pCDH-NC).Western Blot and qRT-PCR validate the success of the overexpression model at the protein and gene levels,respectively.CCK-8 assay was used to detect the effect of overexpression of CLEC-2 on the proliferation of MV4-11 cells.Flow cytometry was used to detect the cycle distribution and apoptosis of MV4-11 cells.Using the KEGG database analysis of the downstream regulatory pathway of the CLEC-2 gene,Western Blot verified the expression of the corresponding protein downstream of the pathway.Results:(1)Bioinformatics analysis showed that the expression level of CLEC-2 in AML cells was lower than that of normal granulocytes.(2)The qRT-PCR results showed that the expression of CLEC-2 in newly diagnosed AML patients was significantly lower than that in normal control group,and there was no significant difference in the expression of CLEC-2 between acute lymphoblastic leukemia patients and normal control group.The expression level of CLEC-2 in AML patients with complete remission after treatment was higher than that of initial diagnosis.Through the correlation analysis between the basic clinical characteristics and CLEC-2 expression level of AML patients,the proportion of patients aged≥60 years in the low CLEC-2 expression group was significantly higher than the high CLEC-2 expression group.The proportion of patients with WBC≥20×10^9/L was significantly higher than the high expression group.ELIS A results showed that the level of CLEC-2 in plasma of newly diagnosed AML patients was significantly lower than that of normal controls.(3)The CLEC-2 overexpressed MV4-11 cell model(pCDH-clec-2)was successfully constructed at mRNA and protein levels.Overexpression CLEC-2 can significantly inhibit the proliferation of MV4-11 cells,and the proportion of cells in G0/G1 phase was increased.After overexpression of CLEC-2,the expression of apoptosis pathway protein Bcl-2 was decreased and the expression of BAX protein was elevated,which promoted the apoptosis of MV4-11 cells.Western Blot results showed that overexpression of CLEC-2 inhibited the expression of phosphorylated ERK protein,hence MAPK/ERK pathway may be involved in the regulation of downstream related mechanisms.Conclusion:(1)The expression of CLEC-2 was lower in newly diagnosed AML patients,and increased in patients with complete remission after treatment compared to before treatment.(2)Overexpression of CLEC-2 can affect the cell cycle distribution,inhibit the proliferation and promote apoptosis of MV4-11 cells.