Increased Expression of RUNX1 in Liver Correlates with NASH Activity Score in Patients with Non-Alcoholic Steatohepatitis (NASH)

Figures

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  Figure 1. Correlation between RUNX1 mRNA expression and histopathological parameters. Expression of RUNX1 mRNA was analyzed by qRT-PCR in liver tissue samples from patients with NASH (n = 43), hepatic steatosis (n = 46) and normal liver tissue (n = 33) and correlated to histopathologic proven (A) NASH activity score (B) steatosis grade (C) inflammation grade and (D) fibrosis grade. HPRT mRNA expression was determined for normalization, statistical differences between several grades were analyzed by Kruskal-Wallis Test (p < 0.05 was considered significant) and ‘r’ denotes the Pearson’s correlation coefficient.

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  Figure 2. Immunohistochemical (IHC) analysis of RUNX1 expression in NASH patients. (A) RUNX1 immunostained images (20× objective) with an increasing number of brown nuclear immuno-positive cells (score 1–4). RUNX1 positivity was mostly observed in the non-parenchymal cells. Hematoxylin stained nuclei were distinguishable from RUNX1-positive brown nuclei. (B) Correlation between RUNX1 IHC and NASH activity score (n = 16), (C) RUNX1 IHC score and fibrosis grade (n = 16), and (D) RUNX1 IHC score and inflammatory grade (n = 16) in NASH patients. (E) Correlation between RUNX1 mRNA and its IHC score in liver tissues of patients (n = 16). The Pearson correlation (r) and statistical significance (p) were calculated.

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  Figure 3. RUNX1 and angiogenic gene expression in endothelial cells maintained in conditioned medium (CM) from palmitic acid (PA) treated hepatoma (Huh7) cells. (A) Huh7 cells treated with 0.2 mM PA for 48 h were analyzed for mRNA expression of RUNX1 and genes identified as angiogenesis associated DEGs in human NASH samples. The dotted line represents the control showing gene expression in Huh7 cells treated with BSA (n = 4). (B) HUVECs incubated with CM from PA-Huh7 cells were analyzed for mRNA expression of RUNX1, its target and angiogenic genes. The dotted line represents control, showing gene expression in HUVECs treated with CM from BSA-Huh7 cells (n = 4). 18S RNA expression was used for normalization. (C) Huh7 cells were treated with BSA or PA or CM alone and analyzed for the release of VEGF, PDGF-BB and TGF-β (pg/mL) (n = 3). (D) Relative RUNX1 mRNA expression in HUVECs treated with VEGF and TGF-β (10 ng/mL each) for 24 h. Un-induced cells without any manipulation were used as respective controls (Dotted line) (n = 3). 18S RNA expression was used for normalization. Data represent mean ± SD. * p < 0.05; ** p < 0.001.

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  Figure 4. RUNX1 alters expression of angiogenic and adhesion molecules in endothelial cells (A) HUVECs, treated with RUNX1 siRNA or NC siRNA and incubated with CM from PA-Huh7 cells, were analyzed for mRNA expression (fold change) of angiogenic, adhesion molecule and RUNX1 target genes (n = 3). (B) HUVECs transfected with RUNX1 expression plasmid (pRUNX1), control plasmid (pControl, i.e., empty vector) and/or incubated with VEGF (10 ng/mL) were analyzed for mRNA expression of adhesion molecule and chemotactic genes. HUVECs without any treatment were used as respective controls (Dotted line) (n = 3). 18S RNA expression was used for normalization. (C) Quantitative analysis of flow cytometry from (C) is shown (n = 3). (D) CCL2 levels (pg/mL) in culture media of HUVECs transfected with RUNX1 expression plasmid (pRUNX1), control plasmid (pControl, empty vector) and/or incubated with VEGF (10 ng/mL) (n = 4). Data represent mean ± SD. * p < 0.05 and ** p < 0.001.

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  Figure 5. RUNX1 enhances the angiogenic activitiy of endothelial cells. (A) Representative tube formation images of HUVECs on matrigel (4× objective) transfected with RUNX1 expression plasmid (pRUNX1), control plasmid (pControl, empty vector) and/or incubated with VEGF (10 ng/mL). (B) Average number of branch points per field and (C) tube length per field formed by HUVECs on matrigel under conditions described in (A) (n = 3). Data represent mean ± SD. * p < 0.05.