Diagnostic and prognostic potential of eight whole blood microRNAs for equine sarcoid disease

Abstract

MicroRNAs have been proposed as biomarkers for equine sarcoids, the most prevalent equine skin tumors globally. This study served to validate the diagnostic and prognostic potential of whole blood microRNAs identified in a previous study for long-term equine sarcoid diagnosis and outcome prediction. Based on findings of a clinical examination at the age of 3 years and a follow-up following a further 5–12 years, 32 Franches-Montagnes and 45 Swiss Warmblood horses were assigned to four groups: horses with regression (n = 19), progression (n = 9), new occurrences of sarcoid lesions (n = 19) and tumor-free control horses (n = 30). The expression levels for eight microRNAs (eca-miR-127, eca-miR-432, eca-miR-24, eca-miR-125a-5p, eca-miR-134, eca-miR-379, eca-miR-381, eca-miR-382) were analyzed through reverse transcription quantitative polymerase chain reaction in whole blood samples collected on initial examination. Associations of sex, breed, diagnosis, and prognosis with microRNA expression levels were examined using multivariable analysis of variance. Sex and breed influenced the expression level of five and two microRNAs, respectively. Eca-miR-127 allowed discrimination between sarcoid-affected and tumor-free horses. No variation in microRNA expression was found when comparing horses with sarcoid regression and progression. Expression levels of eca-miR-125a-5p and eca-miR-432 varied in male horses that developed sarcoids throughout the study period in comparison to male control horses. While none of the investigated miRNAs was validated for predicting the prognosis of sarcoid regression / progression within young horses with this condition, two miRNAs demonstrated potential to predict if young male (though not female) tumor-free horse can develop sarcoids within the following years. Sex- and breed- biased miRNAs exist within the equine species and have an impact on biomarker discovery.

The potential of three whole blood microRNAs to predict outcome and monitor treatment response in sarcoid-bearing equids

“…Eca-miR-127, eca-miR-379, and eca-miR-432 were chosen as candidate miRNAs based on their potential prognostic and/ or diagnostic properties shown in two previous studies (Unger et al 2019b ; Cosandey et al 2021 ). Eca-miR-30d was selected as an endogenous control and cel-miR-39-3p as an exogenous control as previously recommended (Cosandey et al 2021 ). Information on miRNA primers and probes have been published elsewhere (Cosandey et al 2021 ).…”

“…Eca-miR-30d was selected as an endogenous control and cel-miR-39-3p as an exogenous control as previously recommended (Cosandey et al 2021 ). Information on miRNA primers and probes have been published elsewhere (Cosandey et al 2021 ). The RT- and qPCR reactions were performed as previously described.…”

“…We have previously proposed selected serum and whole blood miRNAs to provide a non-invasive method to diagnose ES (Unger et al 2019a , 2019b , 2021 ; Cosandey et al 2021 ). However, during the validation phase, we identified multiple factors that can bias equine circulating miRNA expression, most importantly biological variables such as breed, sex, and age, and in the case of serum samples additional pre-analytical factors such as hemolysis (Unger et al 2019a , 2021 ; Cosandey et al 2021 ).…”

“…We have previously proposed selected serum and whole blood miRNAs to provide a non-invasive method to diagnose ES (Unger et al 2019a , 2019b , 2021 ; Cosandey et al 2021 ). However, during the validation phase, we identified multiple factors that can bias equine circulating miRNA expression, most importantly biological variables such as breed, sex, and age, and in the case of serum samples additional pre-analytical factors such as hemolysis (Unger et al 2019a , 2021 ; Cosandey et al 2021 ). In whole blood, for example, male equids had higher miRNA levels compared to female equids and some miRNA biomarker candidates were breed- or sex-specific, which limits their use as universally applicable diagnostic tools for ES in equine practice (Cosandey et al 2021 ).…”

“…The goal of this study was to assess the diagnostic and prognostic properties of three whole blood miRNAs that have recently been suggested as biomarkers for ES disease in an additional independent study cohort (Unger et al 2019b ; Cosandey et al 2021 ). Eca-miR-127, eca-miR-379, and eca-miR-432, are all encoded by a large miRNA cluster on equine chromosome 24, which corresponds to the well-documented, predominantly tumor-suppressive human 14q32 miRNA cluster, and has previously been proposed to play a role in ES pathogenesis (Zehavi et al 2012 ; Bogedale et al 2019 ; Unger et al 2019b ).…”