lncRNA NORAD is consistently detected in breastmilk exosomes and its expression is downregulated in mothers of preterm infants

Abstract

Breast milk is the ideal food for infants and undoubtedly has immediate and long-term benefits. Breast milk contains extracellular vesicles (EVs) i.e., exosomes secreted by maternal breast cells. Exosomes carry genetic material, such as long non-coding RNAs (lncRNAs), which possibly participate in cell-to-cell communications, as they are known to regulate critical gene pathways. The aim of the present study was to screen human breastmilk exosomes for their lncRNA cargo and to examine exosomal lncRNA levels associated with milk obtained from mothers that gave birth at term or prematurely (<37 weeks of gestation). Samples were collected at 3 weeks postpartum from 20 healthy, breastfeeding mothers; 10 mothers had given birth at full-term and 10 mothers preterm. Exosomal RNA was extracted from all samples and the expression of 88 distinct lncRNAs was determined using reverse transcription-quantitative PCR. A total of 13 lncRNAs were detected in ≥85% of the samples, while 31 were detected in ≥50% of the samples. Differential expression analysis of the lncRNAs between the two groups revealed ≥2-fold differences, with generally higher lncRNA concentrations found in the milk of the mothers that gave birth at term compared with those that gave birth preterm. Among these, the non-coding RNA activated at dNA damage (NORAD) was prominently detected in both groups, and its expression was significantly downregulated in the breast milk exosomes of mothers who delivered preterm. On the whole, the present study demonstrates that breast milk lncRNAs may be important factors of normal early human development. collectively, the presence of lncRNAs in human breast milk may explain the consistent inability of researchers to fully 'humanize' animal milk.

Human Milk Extracellular Vesicles: A Biological System with Clinical Implications

“…CRNDE is a metabolic regulator that promotes aerobic glycolysis [ 183 ], while LINC00657 helps maintain genomic stability under stress [ 184 , 185 ]. Alterations of these lncRNAs in hMEVs are consistent with adaptive responses of the preterm infants against hypoxic conditions [ 80 ]. In addition, LINC00657 is one of the most abundant lncRNAs in hMEVs, and its decrease may serve as a biomarker of perinatal hypoxic stress [ 80 ].…”

“…By high-throughput analyses, up to 920 proteins [ 72 ], 1523 miRNAs [ 73 ], and 395 lipids [ 74 , 75 ] have been identified in hMEVs. The expression and amount of hMEV molecular components vary depending on the maternal physiological and pathological states [ 76 , 77 , 78 , 79 , 80 , 81 , 82 ]. Nonetheless, some of the functional molecules are highly abundant and frequently identified in hMEV studies.…”

“…Preterm delivery is associated with differences in functional cargos of hMEVs. Mourtzi et al [ 80 ] compared hMEVs and their lncRNA levels in mothers of term (≥37 weeks of gestation) vs. preterm (<37 weeks of gestation) birth (n = 10 each group). Of the 31 hMEV-derived lncRNAs measured, 4 lncRNAs differed significantly in milks of the preterm group; LRRC75A-AS1 was higher, and CTC-444N24.11, CRNDE and LINC00657 (NORAD; non-coding RNA activated at DNA damage) were lower.…”

Extracellular vesicles in human milk

“…The main lncRNAs are colorectal neoplasia differentially expressed (CRNDE), differentiation antagonizing nonprotein coding RNA (DANCR), growth arrest-specific 5 (GAS5), steroid receptor RNA activator 1 (SRA1) and ZNFX1 antisense RNA1 (ZFAS1). The lncRNA NORAD (Noncoding RNA activated at DNA damage) was found to be ubiquitously present in human MDEVs and was significantly downregulated in the MDEVs of mothers who delivered prematurely [32 ▪ ].…”

“…lncRNAs, defined as transcribed RNA molecules greater than 200 nucleotides in length, have been detected in human MDEVs [31,32 ▪ ]. lncRNA interacts with RNA and proteins to regulate gene expression, signal pathways and chromatin function [33].…”