Identification of miRNA148a-3p as a novel non-invasive potential biomarker for bacterial infection-related ACLF

Background miRNAs hold great promise as potential biomarkers in liver diseases including acute-on-chronic liver failure (ACLF). Bacterial infection (BI) is a common precipitant of ACLF in humans. Herein, we aim to identify putative miRNA biomarkers in a novel bacterial infection-related acute-on-chronic liver injury model (BI-ACLI), mimicking ALCF conditions with LPS injection in a knock-out mouse with chronic hepatobiliary injury (Abcb4-/-).

Methods Fifteen-week-old C57BL/6J (wild-type) or Abcb4-/- (knock-out) mice (N = 16 each) were treated with IP injections of either LPS (4 mg/kg) or sterile saline solution (0.9 % NaCl). Transcriptomics were characterized by RNA sequencing (RNA-Seq) of mRNA libraries constructed from liver samples of three randomly selected male mice from each group (NebNext Ultra II RNA library preparation kit) using NextSeq500 (Illumina). Liver-specific steady-state mRNA levels (relative to Gapdh) of several differentially expressed genes (DEGs) identified by RNA-seq were confirmed by Taqman assays (Applied Biosystems). Screening of differentially expressed miRNAs was performed in the same samples by miScript miRNA PCR arrays (Qiagen). For mouse-to-human translation, identified miRNAs in mouse livers were further quantified in plasma samples of healthy controls and patients with chronic liver diseases (CLD) with or without bacterial infection.

Results Trabscriptomic data revealed 145 DEGs, which were significantly up- (N = 127) or down-regulated (N = 18) in KO-LPS group compared to their counterparts. Differential expressions of identified DEGs were found to be in line with RT-PCR results for a number of genes tested, including Rantes, IL-22, IL-2 and IL-6. Seven out of 84 miRNAs screened were shown to be differentially expressed in ACLI mice (KO-LPS), namely let-7c-5p, let7b-5p, mir148a-3p, mir21a-5p, mir23a-3p, mir25-3p, and mir29c-3p. Of these seven miRNAs, mir148a-3p was further confirmed to be significantly elevated in CLD patients with current BI (time of BI detection  < 2 weeks, N = 14), compared to those without BI (p=0.038, N = 15), with past BI (time of BI detection  >  1 year, N = 16) (p=0.043) and healthy controls (p < 0.01, N = 7).

Conclusions Differential expression of hepatic cytokines, chemokines and miRNAs in Abcb4-/- mice upon LPS challenge provide hints for identifying potential biomarkers in this dual-hit model. Our in vivo and human findings suggest that circulating mir148a-3p might be a potential marker for BI-related ACLF.

Publication History

Article published online:
04 January 2021