Fig 1: DCN secreted by CAFs is gradually downregulated during HCC progression. (A) Venn diagram of DEGs enriched in ECM-related pathways between public dataset GSE77509 and TCGA. (B–D) Relative mRNA expression of DCN among normal tissue, tumor tissue, and PVTT tissue in the GEO datasets GSE69164 (B), GSE74656 (C), and our dataset (D). (E) DCN protein expression in normal tissue (N), tumor tissue (T), and PVTT (P) tissue as obtained by immunoblot analysis. (F,G) Co-immunofluorescence staining of DCN with E-cadherin (F) and DCN with a-SMA (G) was performed in normal tissue, tumor tissue, and PVTT tissue. Scale bar, 100 µm. a-SMA, alpha-smooth muscle actin; PVTT, portal vein tumor thrombosis. TCGA, The Cancer Genome Atlas. Data presented as mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001, Student’s t-test.
Fig 2: DCN inhibits migration and invasion of HCC cell lines. (A,B) Wound-healing assays for HCCLM3 (A), and Hep3B (B) cells treated with DCN (1 µg/mL) or the negative control. Scale bars, 200 µm. (C,D) Transwell assays for HCCLM3 (C), and Hep3B (D) cells treated with DCN(1 µg/mL) or the negative control. Scale bars, 100 µm. Data presented as mean ± SEM. **P < 0.01, Student’s t-test.
Fig 3: DCN downregulates integrin ß1 expression. (A) Western blot analysis of integrins expression in HCCLM3 and Hep3B cells treated with DCN (1 µg/mL) or the negative control. (B) qPCR analysis of integrin a5 expression in HCCLM3 and Hep3B cells treated with DCN (1 µg/mL) or the negative control. (C) qPCR analysis of integrin ß3 expression in HCCLM3 and Hep3B cells treated with DCN (1 µg/mL) or the negative control. (D) Representative images of integrin ß1 and DCN expressions in normal tissue, tumor tissue, and PVTT tissue obtained by co-immunofluorescence staining. Scale bar, 100 µm. ITGA5, integrin a5; ITGB3, integrin ß3; PVTT, portal vein tumor thrombosis. Data presented as mean ± SEM. ***P < 0.001, Student’s t-test.
Fig 4: DCN treatment combined with integrin ß1 downregulation synergistically inhibits migration and invasion of HCC cell lines. (A,B) Wound-healing assays using integrin ß1 knocked-down or negative control HCCLM3 (A), and Hep3B (B) cells treated with DCN (1 µg/mL) or the negative control. Scale bars, 200 µm. (C,D) Transwell assays using integrin ß1 knocked-down or negative control HCCLM3 (C), and Hep3B (D) cells treated with the addition of DCN (1 µg/mL) or the negative control. (E) Western blot analysis of integrin ß1 expression using integrin ß1 knocked-down or negative control HCCLM3 and Hep3B cells treated with DCN (1 µg/mL) or the negative control. (F) Co-IP assay of DCN with integrin ß1, as detected by immunoblot analysis. Scale bars, 100 µm. ITGB1, integrin ß1. Data presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t-test.
Fig 5: Spironolactone inhibits CNV through induction of anti-angiogenic decorin protein. a On western blot, the decorin (DCN) level decreases in the rat retinal pigment epithelium (RPE)-choroid at different time point (day 1, 3, 7, and 10) after laser induction compared to the normal rat RPE-choroid (ctrl). Densitometric quantification shows significant decrease of DCN protein in rat RPE-choroid at day 3 after laser induction. b Intravitreal injection (IVT) of recombinant mDCN protein in rat eyes inhibits choroidal vascular leakage on fluorescein angiography (FA); DCN 10 µg/ml significantly reduces the CNV angiographic grades (p = 0.02), whereas DCN at both 1 µg/ml (p = 0.0461) and 10 µg/ml (p = 0.0388) decrease the size of CNV induced by laser. c IVT of DCN siRNA in rat eyes with laser-induced CNV. Treatment with spironolactone (Spiro) in the presence of control siRNA significantly increases the DCN protein level in the rat RPE-choroid 48 h after laser induction. IVT of DCN siRNA prevents the increase in the DCN protein induced by spironolactone. d Treatment with spironolactone in the presence of control siRNA inhibits choroidal neovascular leakage on FA at day 14 after laser induction (p = 0.0194). IVT of DCN siRNA at day 0 and 3 after laser induction abrogates the effect of spironolactone on vascular leakage (p = 0.0344). Spironolactone in the presence of control siRNA reduces significantly CNV volume as compared to laser control (p < 0.0001). DCN siRNA injected at day 0 and 3 after laser induction abrogates the effect of spironolactone on CNV volume (p = 0.0003). Western blot data are expressed as mean ± SEM. Dots represent individual RPE-choroid sample. Non-parametric Kruskal–Wallis test was used. FA Data are expressed as the incidence of CNV angiographic grades of the total laser impacts in each group. CNV volumes are expressed as mean ± SEM of the average CNV size per rat. n represents the number of rats. Linear mixed model was used for statistical analyses. *p < 0.05, **p < 0.01, ***p < 0.001
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