Figure 7

Involvement of lncRNA-TUG1-miR-34a-5p-VEGF network in angiogenesis for hepatoblastoma. (a) An in vivo hepatoblastoma model was established by injection of HuH-6 cells expressing the non-specific sequences or precursors of miR-34a-5p. Tumor growth was compared between miR-34a-5p-transfected group and Nc group. Tumor volume (V) was calculated using the formula, V=(L × W²) × 0.5 (n=6; Mann–Whitney U-test; P=0.0409 (2w), P=0.0207 (3w), P=0.0199 (4w)). (b) The tumors were weighed immediately after isolation. The tumor weight was plotted between miR-34a-5p-transfected group and Nc group (n=6; Mann–Whitney U-test; P=0.0283). (c and d) CD31 and Ki67 immunofluoresence staining was conducted to detect microvascular density and cancer cell proliferation. A representative image and statistical result was shown ((n=6; Mann–Whitney U-test; P=0.0133 (c), P=0.0206 (d)). Scale bar: 100 μm. (e) HUVECs were cultured in the following media: HUVEC-specific media as positive control; conditioned media (CM) of HuH-6 cells, CM pre-treated with VEGFA antibody; CM of HuH-6 cells in which the endogenous VEGF expression silenced by RNA interference; CM of HuH-6 cells ectopically expressing miR-34a-5p with or without recombinant VEGFA (n=4; Student’s t-test). Tube formation was determined after 24 h of culture under a phase contrast microscope