Digital PCR (dPCR) Quantification of miR-155-5p as a Potential Candidate for a Tissue Biomarker of Inflammation in Rabbits Infected with Lagovirus europaeus/Rabbit Hemorrhagic Disease Virus (RHDV)

Abstract

MicroRNAs (miRNAs, miRs) are a group of small, 17–25 nucleotide, non-coding RNA sequences that, in their mature form, regulate gene expression at the post-transcriptional level. They participate in many physiological and pathological processes in both humans and animals. One such process is viral infection, in which miR-155 participates in innate and adaptive immune responses to a broad range of inflammatory mediators. Recently, the study of microRNA has become an interesting field of research as a potential candidate for biomarkers for various processes and disease. To use miRNAs as potential biomarkers of inflammation in viral diseases of animals and humans, it is necessary to improve their detection and quantification. In a previous study, using reverse transcription real-time quantitative PCR (RT-qPCR), we showed that the expression of ocu-miR-155-5p in liver tissue was significantly higher in rabbits infected with Lagovirus europaeus/Rabbit Hemorrhagic Disease Virus (RHDV) compared to healthy rabbits. The results indicated a role for ocu-miR-155-5p in Lagovirus europaeus/RHDV infection and reflected hepatitis and the impairment/dysfunction of this organ during RHD. MiR-155-5p was, therefore, hypothesized as a potential candidate for a tissue biomarker of inflammation and examined in tissues in Lagovirus europaeus/RHDV infection by dPCR. The objective of the study is the absolute quantification of ocu-miR-155-5p in four tissues (liver, lung, kidney, and spleen) of rabbits infected with Lagovirus europaeus/RHDV by digital PCR, a robust technique for the precise and direct quantification of small amounts of nucleic acids, including miRNAs, without standard curves and external references. The average copy number/µL (copies/µL) of ocu-miRNA-155-5p in rabbits infected with Lagovirus europaeus GI.1a/Rossi in the liver tissue was 12.26 ± 0.14, that in the lung tissue was 48.90 ± 9.23, that in the kidney tissue was 16.92 ± 2.89, and that in the spleen was 25.10 ± 0.90. In contrast, in the tissues of healthy control rabbits, the average number of copies/µL of ocu-miRNA-155-5p was 5.07 ± 1.10 for the liver, 23.52 ± 2.77 for lungs, 8.10 ± 0.86 for kidneys, and 42.12 ± 3.68 for the spleen. The increased expression of ocu-miRNA-155-5p in infected rabbits was demonstrated in the liver (a fold-change of 2.4, p-value = 0.0003), lung (a fold-change of 2.1, p-value = 0.03), and kidneys (a fold-change of 2.1, p-value = 0.01), with a decrease in the spleen (a fold-change of 0.6, p-value = 0.002). In the study of Lagovirus europaeus/RHDV infection and in the context of viral infections, this is the first report that shows the potential use of dPCR for the sensitive and absolute quantification of microRNA-155-5p in tissues during viral infection. We think miR-155-5p may be a potential candidate for a tissue biomarker of inflammation with Lagovirus europaeus/RHDV infection. Our report presents a new path in discovering potential candidates for the tissue biomarkers of inflammation.

MicroRNAs participate in the regulation of apoptosis and oxidative stress-related gene expression in rabbits infected with Lagovirus europaeus GI.1 and GI.2 genotypes

“…Our recent studies indicate the role of miRs as micro-players of great importance in viral infections in humans and animals ( Hukowska-Szematowicz et al, 2020 ; Ostrycharz and Hukowska-Szematowicz, 2022 ; Hukowska-Szematowicz et al, 2023 ). miRs can regulate gene expression related to apoptosis ( Su et al, 2015 ) and oxidative stress ( Banerjee et al, 2017 ; Fioravanti et al, 2022 ) during diseases, including viral ones ( Hukowska-Szematowicz et al, 2020 ; Fioravanti et al, 2022 ; Ostrycharz and Hukowska-Szematowicz, 2022 ), and thus affect the activity status of signaling pathways ( Chen, 2015 ; Sadri Nahand et al, 2021 ).…”

“…Pathological changes include enlargement of the liver, spleen (splenomegaly), and kidneys, as well as hyperemia and pulmonary edema (results in severe failure manifesting as shortness of breath, worsening by death; Abrantes et al, 2012 ; Rocchi and Dagleish, 2018 ). In these organs, inflammatory foci rich in neutrophils and T and B lymphocytes are observed, as well as an increase in the expression of the inflammatory biomarker—miR-155-5p—in the liver, lungs, and kidneys and a decrease in the spleen ( Hukowska-Szematowicz et al, 2023 ). It is known that systemic hemorrhagic diathesis and intravascular coagulation syndrome (DIC) can lead to death in rabbits ( Trzeciak-Ryczek et al, 2015 ).…”