Carbon tetrachloride- (CCl4-) induced fibrosis mouse model

A total of 35 Balb/c male mice, weighting from 18 to 25 g, with the age of 8 weeks were bought from the Laboratory Animal Center of Zhengzhou University. They were maintained in a 12-hour light/dark room at about 24 °C, and were free to get food and water. For the screening of circRNAs, the fibrosis mouse model was induced by CCl₄ as previously described [32]. In brief, 14 mice were randomly divided into 2 groups, the control group (n=7) and the CCl₄ group (n=7). The CCl₄ solution (v/v, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in olive oil with a final concentration of 5%. Then the mice in the CCl₄ group were intraperitoneally injected with CCl₄ at a dose of 0.3ml/kg 3 times a week for 6 weeks. The mice in the control group were intraperitoneally injected with the equal amount of olive oil alone. For the in vivo experiment, 21 CCl₄-induced mice were randomly assigned into 3 groups, the CCl₄ group (n=7), the CCl₄+shRNA group (n=7), and the CCl₄+shRNA-circ-PWWP2A group (n=7). After 2 weeks of CCl₄ injection, mice in the CCl₄+shRNA-circ-PWWP2A group were intravenously injected via the tail vein with the shRNA-circ-PWWP2A at a dose of 300 μl viruses/mouse (10¹² genome copy particle/ml). Mice in the CCl₄+shRNA group were injected with the negative control adeno-associated virus. After 6-week induction, mice were sacrificed and liver tissues were harvested for testing.