Project description:RNA splicing is an essential step for expression of genes commonly altered in multiple liver diseases. However, how the spliceosomal components participate in the pathogenesis of acute liver failure (ALF) remains poorly defined. Here, we report that KH-type splicing regulatory protein (KSRP) which is downregulated in ALF, regulates RNA splicing in the liver through interacting with the Splicing factor 3b subunit 1 (SF3B1). Overexpression of KSRP resulted in marked amelioration of hepatic injury in vivo. Transcriptomic analysis reveals that KSRP depletion led to severe splicing defects in genes with significant intron retention. Such defects cause substantial loss of proliferation and apoptosis related genes such as EGFR, which further exaggerated liver injury. KSRP promotes RNA splicing of splicing related genes, including SF3B1, PHF5A, and SF3B4, indicating the existence of a positive feedback loop that regulates RNA splicing. Mechanistically, KSRP directly interacts with SF3B1 and enhances binding of SF3B1 to the branch sites located upstream of the exon. Overall, our findings demonstrate a novel mechanism by which KSPR protects against acute liver injury by promoting RNA splicing through interacting with the SF3B complex.
Project description:To investigate the differences in microRNA expression profiles between fibrotic and normal livers, we performed microRNA microarrays for total RNA extracts isolated from mouse livers treated with carbontetrachloride (CCl4) or corn-oil for 10 weeks (n=3/group). MicroRNAs were considered to have significant differences in expression level when the expression difference showed more than two-fold change between the experimental and control groups at p<0.05. We found that 12 miRNAs were differentially expressed in CCl4-induced fibrotic liver. To induce chronic liver fibrosis, seven-week-old mice received 0.6 ml/kg body weight of carbon-tetrachloride (CCl4) dissolved in corn-oil by intraperitoneal (i.p.) injection, twice a week for 10 weeks (n=3). As a control, same number of mice was injected with equal volume of corn-oil for 10 weeks.
Project description:To investigate the altered gene expression levels in mouse fibrotic liver tissues, C57BL6/J mice were intraperitoneally injected with CCl4 or vehicle twice every week. After 8 weeks, livers were harvested and RNA was extracted by Trizol. The gene expression levels were analyzed and compared between CCl4 treated group and vehicle treated (control) group.
Project description:To understand the fibrotic response in the CCl4 induced liver fibrosis model, we performed RNA-seq of liver samples from mice treated with oil or CCl4.
Project description:Liver samples were taken from mice with WT p53 or liver-specific loss of p53 at 8 hrs after treatment with the potent hepatotoxin CCl4 in order to analyse differences in p53-dependent gene expression during the early response to CCl4.
Project description:Five-week old male C57BL/6J mice (20 ± 2 g) were intraperitoneally injected with of 20% CCl4 solution in sterile mineral oil at a dose of 2.5 ml CCl4 per kilogram body weight twice per week for eight weeks. Afterwards, the primary hepatocytes were isolated by pronase/collagenase perfusion digestion followed by subsequent density gradient centrifugation. The isolated primary hepatocytes were counted with a hemocytometer to determine the number and percentage of viable cells using the trypan blue method. The isolated cells with the viability above 90% were used for the experiments.
Project description:Since xenobiotics enter the organism via the liver, hepatocytes must cope with numerous perturbations. In particular, exposure to hepatotoxic agents resulting in liver injury triggers a strong alteration in gene expression. The contribution of these transcriptional regulatory networks to the propagation of injury (i.e. by modulation of metabolic pathways, inflammation, proliferation) are not fully understood. Therefore, we conducted a time-resolved gene array study on mouse liver after exposure to a single intraperitoneal injection of the hepatotoxic compound carbon tetrachloride (CCl4). This model induces a transient injury which reaches its maximum between 2 to 3 days after injection, followed by a precise regenerative response. The experiment included early time points (i.e. 2 and 8h) and late time points (1 to 16 days) to cover the initial events induced by CCl4 metabolization and cell stress, and the regenerative phase between 2-6 days after intoxicaiton. C57BL/6N male mice (8-12 weeks old) were obtained from Charles Rivers (Sulzfeld, Germany). The local Ethics committee approved the experiments. 4-5 mice were used for each time point. Mice received a single intraperitoneal injection of CCl4 (1.6 g/kg body weight) dissolved in 0.5 ml olive oil. The liver tissue was collected after 2h, 8h, and 1, 2, 4, 6, 8 and 16 days following CCl4 administration. As control, mice received 0.5 ml of olive oil intraperitonealy for 1 or 8 days. Healthy liver was collected from untreated mice. At the indicated time points, mice were anesthetized and blood was collected from the heart with a 25 gauge needle, using EDTA as anticoagulant, for analysis of the liver transaminases GOT and GPT in serum. Afterwards, the liver was resected, washed in ice cold PBS and sectioned for further analysis. Liver tissue sections of about 0.5 cm2 were snap frozen in liquid nitrogen and stored at -80ᄚC for subsequent isolation of proteins or RNA. RNA was isolated from mouse liver tissue using the Phenol/Chloroform method (Trizolᆴ, Qiagen, Hilden, Germany) according to the manufacturerメs instructions. RNA concentration and integrity were determined spectrophotometrically in a Nanodropᆴ2000 (ThermoScientific, Waltham MA, USA) and in a Bioanalyzerᆴ (Agilent, Waldbronn, Germany), respectively.
Project description:loss of Hmgb2 in vivo in mouse liver largely prevented CCl4-induced liver fibrosis. The goal of the RNA-seq is to identify the Hmgb2 regulated genes in CCL4 induced liver fibrosis.