Y. Zhou1, G. E. Besner1 1Nationwide Children’s Hospital,Department Of Pediatric Surgery,Columbus, OH, USA
Introduction: Necrotizing enterocolitis (NEC) is the leading cause of death in premature babies. The early diagnosis and differentiation of NEC from neonatal sepsis and of medical NEC from surgical NEC is critical, but has been challenging. Little progress has been made in discovering novel diagnostic and prognostic biomarkers for NEC. Exosomes shed by producer cells and released into bodily fluids (e.g. blood, urine), represent an active process of cell-to-cell communication within the body. They contain a complex mixture of microRNAs, messenger RNAs and proteins from the cell of origin, making them an ideal source for biomarker discovery and diagnostic development. Our goal was to profile the microRNA content of serum exosomes from patients with NEC in an attempt to distinguish them from patients with sepsis, and to distinguish medical from surgical NEC.
Methods: Gestational age and post-conceptual age-matched premature babies were divided into four groups [prematures without acute disease, non-NEC sepsis, medial-NEC (patients who recovered without surgery), and surgical-NEC (patients requiring surgery)]. 400 μl of pooled serum (4 patients/group; 100 μl/patient) was obtained from patients upon the initial development of symptoms. Serum exosomes were isolated and microRNA profiling performed on the circulating exosomes using a Human miRnome miR PCR Array. Differentially expressed microRNAs were confirmed and/or further evaluated by qRT-PCR of exosomal RNA from the same individuals, and from three additional different individuals with the same diagnosis.
Results: Isolated exosomes from patient serum were bi-membrane vesicles, 30-200 nm in diameter, and positive for the exosome markers CD63 and flotillin-1. Microarray analysis revealed significant alterations in the expression of hundreds of microRNAs that had expression levels up- or down-regulated more than two-fold. We found that patients with NEC had significant up-regulation of miR-106, miR-1245a, and miR-224, and down-regulation of miR-145, miR-192, Let-7a, and miR-146a, consistent with previous reports in patients with intestinal ischemia or inflammation. In addition, miR-106 and Let-7a are known to target mRNAs that encode the components of inflammatory or anti-inflammatory signaling pathways including nuclear factor-kappaB (NF-κB) and Interleukin-10. Furthermore, exosomal microRNAs that have not yet been reported as being altered during NEC emerged as potentially novel disease markers, including up-regulation of miR-1323 and miR-524 and down-regulation of miR-215 and miR-19a.
Conclusion: Dynamic changes occur in the microRNA content of circulating exosomes from NEC patients. Serum exosome profiling may identify discriminating microRNA signatures distinguishing non-NEC sepsis from medical-NEC, and for risk stratification for NEC progression and severity. Identification of a panel of microRNAs in circulating exosomes may allow the discovery of biomarkers that signal NEC development.