Figure 8. Administration of Ad-miR-223 attenuates PD-1/PD-L1 activation and tumor angiogenesis, hypoxia in DEN+CCl₄-induced HCC.

C57BL/6J mice were treated with DEN+CCl₄ as described in Figure 1, and were treated with Ad-miR-223 or Ad-GFP. (A) Gross images of HCC tumor masses from Ad-miR-223 and control Ad-GFP-treated mice are shown. (B) The largest tumor diameter and the number of tumor masses between Ad-GFP and Ad-miR-223-treated mice were analyzed. (C, D) Representative immunofluorescence staining of CD31/PCNA (panel C) or hypoxia marker (CA9)/proliferative marker (Ki-67) (panel D) in tumor regions of Ad-GFP and Ad-miR-223-treated liver samples are shown. Relative CD31 and CA9 intensity, the percentages of PCNA and Ki67 positive cells were quantified and are shown in Supporting Figure 12B. (E, F) Protein levels of phosphorylated Tie-2 and VEGFR2 (p-TIE-2, p-VEGFR2), HIF-1α, PCNA and AFP were determined and quantified by western blot. Values represent means ± SEM. *P< 0.05, **P< 0.01, ***P< 0.001.