Backgrounds: Angiogenesis plays an important role in the development of liver fibrosis and portal hypertension. Long non-coding RNAs (lncRNAs) are involved in the liver diseases and angiogenesis.
Objective: This study aimed to investigate the effect of lncRNA-COX-2 in the progress of liver fibrosis, and the underlying mechanisms were also studied.
Methods: Thirty-six SPF Bal B/C mice were randomly assigned into 3 groups with 12 mice in each group. The control group received injection of normal saline (100 µl, twice a week); the CCl4-2M group given intraperitoneal injection of carbon tetrachloride (CCl4) for 2 months; the CCl4-3M group given intraperitoneal injection of CCl4 for 3 months. The hepatic fibrosis was assessed by the hepatic fibrotic areas. Quantitative real-time PCR was performed to evaluate the expressions of lncRNA-COX-2 and COX-2 in the liver tissue, and immunohistochemistry staining was used to detect COX-2 in the liver tissue. Correlation of lncRNA-COX-2 and COX-2 with liver fibrotic areas was completed by using Pearson correlation analysis. In vitro, the EOMA endothelial cells were treated with lipopolysaccharide (LPS) for 6 hours, and lncRNA-COX-2, COX-2, VEGF and VEGFR-2 were evaluated by quantitative real-time PCR and immunohistochemistry staining.
Results:
Extensive nodular formation was observed in the liver after the treatment with CCl4. Compared with the control group, fibrotic areas of liver tissues in the CCl4-2M group and CCl4-3M group were increased by 4 times and 9 times, respectively. The CD31 positive areas and vascular areas were significantly increased in cirrhotic livers. Additionally, up-regulations of COX-2 mRNA and COX-2 protein were observed in both the CCl4-2M group and the CCl4-3M group, compared with the control group. Moreover, the level of lncRNA-COX-2 in the CCl4-2M group and CCl4-3M group were nearly 2 times higher than that in the control group. Meanwhile, the expression of COX-2 and lncRNA-COX-2 were positively correlated with the liver fibrotic areas. (R=0.801, P<0.05; R=0.593, P<0.05). Besides, a positive correlation between the expression of COX-2 mRNA and lncRNA-COX-2 was displayed in cirrhotic liver (R=0.667, P<0.05). And there also was a positive correlation between the CD31 positive areas and lncRNA-COX-2 in cirrhotic liver (R=0.605, P<0.05). In vitro, the gene expression of lncRNA-COX-2, COX-2, VEGF and VEGFR-2 were remarkably increased in LPS-treated EOMA cells. Consistently, the protein expression of COX-2 and VEGF were also obviously increased in LPS-treated EOMA cells.
Conclusions: Expressions of COX-2 and lncRNA-COX-2 increase in the progress of liver fibrosis. LncRNA-COX-2 may exacerbate liver fibrosis via increasing the inhtraheaptic angiogenesis. And the neoangiogenic effect of lncRNA-COX-2 may attribute to its up-regulation of COX-2, VEGF and VEGFR-2.
