Diagnostic potential of three serum microRNAs as biomarkers for equine sarcoid disease in horses and donkeys

Abstract

Background: MicroRNAs (miRNAs) are potential biomarkers for equine sarcoids (ES).

Objectives: To assess eca-miR-331, eca-miR-100, and eca-miR-1 as serum biomarkers for ES disease.

Animals: Sixty-eight ES cases (56 horses, 12 donkeys), 69 tumor-free controls (60 horses, 9 donkeys), and 20 horses with other skin tumors.

Methods: For this case-control study, expression of serum eca-miR-331, eca-miR-100, and eca-miR-1 in ES-affected equids was compared to tumor-free age-, sex-, and breed-matched control horses and donkeys with other skin tumors using reverse transcription quantitative PCR (polymerase chain reaction) for relative miRNA quantification. Biological, preanalytical, and clinical variable influences on miRNA expression were examined. Receiver operator characteristic (ROC) curve analyses were used to determine differences in miRNA expression between groups.

Results: The expression of eca-miR-100 was affected by age (P = .003) and expression of eca-miR-100 and eca-miR-1 were affected by hemolysis (both P < .001). Eca-miR-331 was unaffected by biological variation, hemolysis, ES type, and disease severity. Eca-miR-331 concentrations were higher in ES-affected compared to tumor-free controls (P = .002). The ROC curve analysis indicated an area under the curve of 0.65 (P = .002) with a sensitivity of 60%, specificity of 71%, and positive and negative likelihood ratios of 2.1 and 0.56, respectively, to diagnose ES. Eca-miR-331 expression did not discriminate between horses with ES and other skin tumors. Expression of eca-miR-100 and eca-miR-1 was not different between groups.

Conclusions and clinical importance: Serum eca-miR-331 expression is neither sensitive nor specific enough as a single ES biomarker. If combined with other miRNAs, it may be helpful for ES diagnosis.

Keywords: circulating microRNA; eca-miR-331; equine tumor marker; transcriptomic biomarker.

FIGURE 1

Dot plots depicting relative serum expression of eca‐miR‐331 in tumor‐free control equids and equine sarcoid affected equids. The y‐axis shows the 2^−ΔΔCq values of eca‐miR‐331 on a log scale. The horizontal lines represent the geometric mean. Asterisk represents the significant P value. Cq, cycle quantification

FIGURE 2

Dot plots depicting relative serum expression of eca‐miR‐331 in tumor‐free control horses, horses with other skin tumors, and equine sarcoid affected horses. The y‐axis shows the 2^−ΔΔCq values of eca‐miR‐331 on a log scale. The horizontal lines represent the geometric mean. Asterisk represents the significant P value. Cq, cycle quantification