Figure 4. Knockdown of endogenous Fut1 expression in the liver of an immunocompetent HCC mouse model attenuates hepatocarcinogenesis.

(A) Schematic representation of the hydrodynamic tail vein injection (HTVI) model in C57BL/6 mice. (B) qPCR analysis of Fut1 expression in mice that received HTVI of either empty vector control (EV CTRL) or NRAS, AKT, and sleeping beauty (SB) transposase for HCC induction with samples collected at various time points. n = 22 in total (EV, n = 3; 1 week, n = 3; 2 weeks, n =3; 3 weeks, n = 3; 4 weeks, n = 3; 5 weeks, n = 3; endpoint, n = 7) (1-way ANOVA). (C) Strategy for testing the functional significance of Fut1 in hepatocarcinogenesis. NRAS, AKT, and SB transposase were delivered by HTVI for HCC induction. Lentiviral particles with nontargeting control shRNA (shNTC) or shFut1 were administered twice at 4 and 5.5 weeks. Mice were sacrificed at 10 weeks after plasmid injection. (D) Representative images of dissected livers at the end of the experiment. Scale bars: 5 cm and 2 cm (enlarged images). Successful Fut1 knockdown confirmed by qPCR. n = 11–12 per group. (E) An ex vivo limiting-dilution assay of HCC tumor cells harvested from HTVI mouse models found that the frequency of tumor-initiating cells decreased in the mice administered shFut1 lentiviral particles. The data shown are representative of 3 independent experiments (pairwise tests for differences in stem cell frequencies). *P < 0.05.