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.
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.
Posted by: Shihang Tang, student, West China Hospital, Sichuan University, , China (30-Nov-2016)