Delta-like ligand 4 in hepatocellular carcinoma intrinsically promotes tumour growth and suppresses hepatitis B virus replication

Figure 1 Delta-like ligand 4 expression promotes hepatitis B virus-associated hepatocellular carcinoma tumour growth in vivo. A: Western blot analyses of DLL4 expression in HepG2.2.15 stably transfected with shDLL4 or control vector. GAPDH was used as the loading control. B-D: HepG2.2.15 transfected with shDLL4 or control vector were subcutaneously injected into athymic nude mice (B) (1 × 10⁷ cells per mouse, n = 4-6). Tumour volume (C) and tumour weights (D) are shown. At 18 d and 30 d after implantation, tumours were collected and analysed for DLL4, cleaved Notch1, VEGFA, and VEGFR2 by western blot. Beta-actin was used for the loading control. The blots cropped from different parts of the same gel (E). Band intensities from (E) were measured and the results are presented as the mean ± SD of three independent experiments (F). ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.

Figure 2 Suppression of Delta-like ligand 4 reduces tumour proliferation and neovasculature at the initiation stage of implantation. A and B: Immunohistochemical staining of CD31 (a-d) and Ki67 (e-h) shows mouse neovessels and tumour cell proliferation in paraffin sections of tumour xenografts, respectively. Five fields of each section were quantified for the amount of CD31 staining (C), and the percentage of Ki67 positive cells (D) in tumours transfected with shDLL4 or control vector at 18 d and 30 d after implantation. The tumour vasculature was measured after tumour implantation with shDLL4 HepG2.2.15 or control cells at 30 d (E). The data represent the mean ± SD. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.

Figure 3 Suppression of Delta-like ligand 4 enhances hepatitis B virus viral replication (A), HBx mRNA expression (B), and HBs protein expression (C) in vivo. DNA, RNA, and proteins were extracted from tumour xenografts to analyse HBV viral components at 18 d and 30 d after implantation. The mRNA levels of human IFN-alpha (D), IFN-beta (E), and TNF-alpha (F) from tumours transfected with shDLL4 or control vectors were measured by quantitative RT-PCR and normalized to beta-actin mRNA expression. The data are represented by the mean ± SD. ^aP < 0.05; ^bP < 0.01.