Liver and Serum miRNAs in Toxic Liver Fibrosis

Abstract

Toxic chemical exposure is a risk factor for toxic liver injury in the military, chemical plant workers, and the general public. Early indicators of disease would facilitate medical and strategic decisions involving treatment, protective equipment, and/or relocation. Here, we map liver miRNA to their predicted mRNA targets in a rodent model of hepatotoxicity. We report a panel of serum miRNA that may serve as early, blood‐based indicators of toxic liver injury or fibrosis.Rats were orally administered vehicle control or the hepatotoxicant 4, 4′‐methylenedianiline (MDA; 22, 60 or 162 mg/kg) for 1 or 5 consecutive days. Serum and liver tissue were collected at necropsy, 24 hours after the last exposure. Liver sections were stained with hematoxylin and eosin for histology. Liver tissue and serum miRNA expression was determined by next‐generation sequencing for selected doses of MDA. mRNA gene expression in the liver was measured by microarray. Known and novel miRNAs were mapped to the rat genome using miRDeep2 and differential expression was determined by DESeq (FDR<0.05). mRNA changing by at least 1.5 fold were used for the target analysis using Ingenuity Pathway Analysis (IPA) software. Pathway enrichment analysis was also conducted in IPA.MDA poisoning caused moderate liver fibrosis and bile duct hyperplasia after 5 consecutive doses, as shown by histological analysis. One day after the final dosing, we identified 0, 7, and 38 differentially expressed liver miRNA at 22, 60, and 162 mg/kg, respectively. After 5 doses, 0, 126, and 127 liver miRNA were differentially expressed at 22, 60, and 162 mg/kg, respectively. Sixteen miRNAs were modulated in the serum of the rats exposed to 162 mg/kg of MDA. We also identified novel, previously undescribed differentially expressed miRNAs which may play a role in liver injury progression. We predicted mRNA targets of each differentially expressed miRNA. Pathway enrichment analysis of the predicted targets identified pathways involved in the liver fibrosis adverse outcome pathway, including inflammation, collagen accumulation, and stellate cell activation. Many of the modulated mRNAs targeted by these miRNAs have been previously associated with liver fibrosis.In conclusion, we identified liver miRNAs that may target and regulate gene expression contributing to liver fibrosis. The miRNAs in the serum may serve as prognostic indicators of disease progression.Support or Funding InformationDisclaimer: Research was conducted in compliance with the Animal Welfare Act and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.