Fig. 5. The CD74-MIF receptor interaction facilitates the expression of angiogenic factors in CRC cells.

A qRT-PCR analysis of the relative mRNA levels of MIF and CD74 in HCT116 and DLD-1 CRC cells. Normalized to those of RPLP0. Means ± SD of 2 biological replicates with 3 technical replicates each. B, C Expression analysis of the indicated angiogenic genes in HCT116 (B) or DLD-1 (C) cells after siRNA-mediated knockdown of MIF or CD74 for 72 h; a scrambled siRNA served as the control (‘con’). qRT-PCR were normalized to HPRT1 or RPLP0, respectively. Means ± SD of ≥3 technical replicates from two biological replicates. D, F Angiogenic pathway analysis in CD74-deficient DLD-1 cells. 48 h after DLD-1 cells were transfected with an empty control (D) or CD74 overexpression plasmid (F), they were treated with 100 ng/mL recombinant human MIF (rhMIF) for 24 h. Immunoblots of CD74, pERK, and total ERK. Actin and Hsc70, loading controls. E, G Angiogenic marker expression (VEGFA, CXCL8). DLD-1 cells were treated as described in (D, F) followed by qRT-PCR. Expression of the indicated genes was normalized to that of RPLP0. Means ± SD of ≥3 technical replicates. H Representative images of serial sections from two human CRC patients stained for MIF and CD74 with different stabilization of MIF (MIF^high: stabilized, MIF^low: unstabilized) and expression levels of CD74 (CD74^high, CD74^low). Scale bars, 200 µm. I, J Correlation between patients with stabilized MIF (MIF^high) and CD74 levels (CD74^high, CD74^low) (I) and patients with unstabilized MIF (MIF^low) and CD74 levels (CD74^high, CD74^low) (J) on overall survival of patients. Log-rank (Mantel-Cox) test for comparison of indicated groups. A, B, C, E, G p values were calculated with Student’s t tests for comparison of indicated groups; ns = not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.