Effect of ubiquitinated ligase c-Cbl on proliferation and differentiation of acute myeloid leukemia cells

Objective To investigate the effect of ubiquitylated ligase c-Cbl on the proliferation and differentiation of acute myeloid leukemia cell line THP-1.

Methods THP-1 cells stably transfected with HBLV-h-CBL-ZsGreen-PURO knockdown and HBLV-h-CBL-HA-BSD overexpression viral vectors were constructed and screened.Cells were divided into four groups including NC group (c-Cbl knockdown group control), S3 group (c-Cbl knockdown group), EV group (c-Cbl overexpression group control) and c-Cbl group (c-Cbl overexpression group).Cell Counting Kit-8 (CCK-8 kit) and cell cycle and apoptosis detection kit were used to detect the proliferation and apoptosis of the stable cells.The expression of apoptosis-related proteins (Bcl-2, Caspase-9, Cleaved-Caspase-9) in the gene knockdown or overexpression cells was detected by Western blotting.Nitrotetrazolium blue chloride (NBT) reduction test, Wright-Giemsa staining and flow cytometry were used to detect the expression of surface differentiation antigen CD11b.The expression of PU.1 protein was detected by Western blotting to study the differentiation ability of stably transformed cells.

Results After transfection with lentivirus, the levels of c-Cbl gene in S3 group were decreased compared with NC group.Compared with EV group, levels of c-Cbl gene were increased in c-Cbl group.CCK8 and flow cytometry showed that c-Cbl could promote THP-1 proliferation(P < 0.01).Western blotting results showed that c-Cbl could inhibit the expression of apoptotic protein and promote the expression of Bcl-2 protein.The results of NBT reduction test showed that the number of NBT-positive cells increased after c-Cbl knockout, and the number of positive cells decreased after c-Cbl overexpression(P < 0.01).The results of Wright-Giemsa staining showed that c-Cbl knockdown made the nucleus differentiate into kidney type and broad bean type.Flow cytometry showed that CD11b expression increased after c-Cbl knockout, and decreased after overexpression.Western blotting results also showed that the expression of PU.1 protein increased after c-Cbl knockout, but decreased after overexpression.

Conclusions C-Cbl can promote the proliferation of THP-1 cells and inhibit its apoptosis and differentiation.