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CD147 as potential therapeutic target in AML
Results
CD147 is directly involved in hematopoietic progenitor cell proliferation
We analyzed CD147 expression at both mRNA and pro- tein level in CD34+ HPCs and during Mo and G prolifera- tion and differentiation of these cells. CD147 is well expressed in CD34+ HPCs and its level of expression decreases during Mo and G differentiation and maturation of these cells (Figure 1A-C and Online Supplementary Figure
S1A and B). Western blot analysis shows that CD147 pro- tein is highly glycosylated (HG-CD147 40-60 kDa) in CD34+ HPCs and at all stages of Mo and G cell differenti- ation (Figure 1B), indicating the presence of CD147 pro- tein in a stable and biologically active conformation main- ly translocated to plasma membrane,5 as also shown by flow cytometry analysis (Figure 1C and Online Supplementary Figure S1B). To analyze the role of CD147 in HPCs, we performed siRNA-mediated CD147 knock- down experiments in CD34+ HPCs. We transiently trans-
A
Figure 1. CD147 is down-regulated dur- ing monocytic (Mo) and granulocytic (G) differentiation of CD34+ hematopoietic progenitor cells (HPCs) and is involved in HPC proliferation. (A) qRT-PCR analy- sis of CD147 mRNA expression during selective Mo and G proliferation and dif- ferentiation of CD34+ HPCs, as com- pared to U937 and HL-60 leukemic cells. (B) Western blot analysis of CD147 protein expression level in CD34+ cells and Mo and G differentiat- ing HPCs; Jurkat and SKBR3 cells are shown as positive controls of CD147 expression; HG- and LG- are indicated
B for High- and Low- glycosylated CD147 isoforms; actin is shown as an internal control; molecular weights are indicated (kDa). (C) Flow cytometry analysis of CD147 membrane protein expression during Mo and G differentiation and maturation of HPCs. (D) Flow cytometry analysis of CD147 membrane protein expression in Mo and G differentiating transfected (CD147-siRNA)-HPCs, as compared to transfected (c-siRNA)-HPCs of control, at day 2 of Mo and G cultures corresponding to day 3 post transfec- tion. (E) Cell growth inhibition of (CD147-siRNA)-HPCs, as compared to (c-siRNA)-HPCs, grown under both G and Mo liquid culture conditions. (F) Clonogenic assays performed under G and Mo culture conditions with (CD147- siRNA)-HPCs, as compared to (c-siRNA)- HPCs. (A, C-F) Mean±Standard Error of Mean of three independent experi- ments is shown. *P<0.05; **P<0.01; ***P<0.001. (B) One representative experiment out of three is shown. AU: arbitrary units; MFI: mean fluorescence
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