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Updated: May 6, 2026
Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis
Published on: December 9, 2022
Profiling circulating microRNA expression in experimental sepsis using cecal ligation and puncture
Shao-Chun Wu1, Johnson Chia-Shen Yang, Cheng-Shyuan Rau
1Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
Abstract:
The levels of circulating microRNAs (miRNAs) in mice with experimental sepsis induced by cecal ligation and puncture (CLP) were determined using whole blood samples obtained from C57BL/6 mice at 4, 8, and 24 h after CLP; miRNA expression analysis was performed in these samples using an miRNA array. Microarray analysis revealed upregulation of 10 miRNA targets (miR-16, miR-17, miR-20a, miR-20b, miR-26a, miR-26b, miR-106a, miR-106b, miR-195, and miR-451). The expression of these miRNA targets in the whole blood, serum, and white blood cells (WBCs) of CLP mice was quantified using quantitative real-time PCR; these values were compared to those in sham-operated C57BL/6 mice, and the results indicated that these miRNA targets were significantly up-regulated in the whole blood and serum but not in the WBCs. In addition, the levels of these 10 miRNA targets in the serum of Tlr2-/-, Tlr4-/-, and NF-κB-/- mice at 8 h after CLP did not decrease significantly., which indicated that the transcription of these miRNAs was not directly mediated by the TLR2/NF-κB or TLR4/NF-κB pathway, and pathways induced by exposure to the gram-positive or gram-negative bacteria. Immunoprecipitation with the Argonaute 2 ribonucleoprotein complex revealed significantly increased expression of the 10 miRNA targets in the serum of mice after CLP, and the levels of 6 (miR-16, miR-17, miR-20a, miR-20b, miR-26a, and miR-26b) of these 10 miRNA targets increased significantly in exosomes isolated using ExoQuick precipitation solution. In this study, we identified circulating miRNAs that were up-regulated after CLP and determined the increase in the levels of these miRNAs, and our results suggest that circulating Ago2 complexes and exosomes may be responsible for the stability of miRNAs in the serum.
Insights
Circulating microRNAs (miRNAs) are upregulated in sepsis, particularly within serum and exosomes. These miRNAs are not directly regulated by TLR pathways, suggesting alternative stability mechanisms in sepsis models.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection.
- MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and are implicated in various diseases, including sepsis.
- Understanding circulating miRNA profiles can provide insights into sepsis pathogenesis and potential biomarkers.
Purpose of the Study:
- To identify and quantify circulating microRNAs (miRNAs) in a mouse model of experimental sepsis.
- To investigate the expression patterns of specific upregulated miRNAs in different blood components.
- To explore the potential regulatory pathways and mechanisms responsible for miRNA stability in sepsis.
Main Methods:
- Cecal ligation and puncture (CLP) model in C57BL/6 mice to induce experimental sepsis.
- Whole blood, serum, and white blood cell (WBC) samples collected at 4, 8, and 24 hours post-CLP.
- miRNA microarray analysis to identify differentially expressed miRNAs.
- Quantitative real-time PCR (qRT-PCR) to validate miRNA expression.
- Analysis in Toll-like receptor (TLR) knockout mice (Tlr2-/-, Tlr4-/-, NF-κB-/-).
- Argonaute 2 (Ago2) immunoprecipitation and exosome isolation (ExoQuick) to assess miRNA localization and stability.
Main Results:
- Microarray analysis revealed significant upregulation of 10 specific miRNAs (miR-16, miR-17, miR-20a, miR-20b, miR-26a, miR-26b, miR-106a, miR-106b, miR-195, miR-451) in CLP mice.
- These upregulated miRNAs were predominantly found in whole blood and serum, but not significantly in WBCs.
- miRNA expression was not significantly altered in TLR knockout mice, suggesting independence from TLR2/NF-κB or TLR4/NF-κB pathways.
- Increased levels of these miRNAs were detected in circulating Ago2 complexes and, notably, in exosomes isolated from serum.
Conclusions:
- Circulating miRNAs, including a panel of 10 specific targets, are significantly upregulated in experimental sepsis.
- The observed upregulation is primarily in serum and not directly mediated by TLR signaling pathways.
- Circulating Argonaute 2 complexes and exosomes play a crucial role in the stability and circulation of miRNAs during sepsis.
