Animal in vivo study

All the animal protocols were in accordance with the Guidelines for Animal Experiments of Tianjin Medical University Animal Care and Use Committee. Airn knockout (Airn-KO) C57BL/6N mice were generated by CRISPR/Cas9 system (Cyagen, Suzhou China). In brief, genomic DNA was extracted from the Airn-KO mice and was PCR amplified using the primers (F: 5′-AGACACATTTAGTTGGTGGTTGGTCG-3′, R: 5′-TCTTCCACACCCAGGTGGCTTT-3′, R: 5′-AGGAAGTAGGCTCATGGGAGGAG-3′). The product of 800 bp was used for the amplicon and the sequence was confirmed by Sanger sequencing. All wild type (WT) and Airn-KO mice were generated from Airn heterozygous mice, they were kept in a standard 12-h light–dark cycle under the specific pathogen-free conditions with free access to water and food. All liver fibrosis models were performed in male mice unless indicated. For CCl₄-induced liver fibrosis model, twenty WT mice and twenty Airn-KO mice were randomly divided into four groups: WT mice intraperitoneally injected oil (WT, n = 10), WT mice intraperitoneally injected CCl₄ (WT + CCl₄, n = 10), Airn-KO mice intraperitoneally injected oil (Airn-KO, n = 10), and Airn-KO mice intraperitoneally injected CCl₄ (Airn-KO + CCl₄, n = 10). They were administered 5% CCl₄ (v/v) (Sigma-Aldrich, St. Louis, MO, USA) dissolved in oil (0.3 ml/kg body weight) thrice per week for 6 weeks via intraperitoneal injection. For BDL-induced liver fibrosis model, forty WT mice and forty Airn-KO mice were randomly divided into four groups. WT mice were treated with sham operation (WT, n = 20), WT mice were treated with BDL (WT + BDL, n = 20), Airn-KO mice were treated with sham operation (Airn-KO, n = 20) and Airn-KO mice were treated with BDL (Airn-KO + BDL, n = 20). Eighteen days later, all mice were sacrificed under anesthesia with 3% sodium pentobarbital (45 mg/kg, ip). For over-expressed Airn model, adeno-associated virus (AAV8) vectors were used to over-express Airn in mice, and AAV8-GFP was used as a control. Thus, forty Balb/c mice were divided into four groups randomly: Mice were treated with oil in combination with injection of AAV8-GFP (AAV8-GFP, n = 10), CCl₄ in combination with injection of AAV8-GFP (AAV8-GFP + CCl₄, n = 10), oil in combination with injection of AAV8-Airn (AAV8-Airn, n = 10), and CCl₄ in combination with injection of AAV8-Airn (AAV8-Airn + CCl₄, n = 10). Mice in AAV8-GFP + CCl₄ group and AAV8-Airn + CCl₄ group were injected with AAV8-GFP and AAV8-Airn respectively via the tail vein 2 weeks after the first injection of CCl₄, and administered 5% CCl₄ (v/v) dissolved in oil (0.2 ml/kg body weight) thrice per week via intraperitoneal injection. AAV8-GFP and AAV8-Airn group animals were injected with an equivalent volume of oil. After treatment with CCl₄ for 8 weeks, all mice were sacrificed under anesthesia with 3% sodium pentobarbital (45 mg/kg, ip).