DataSheet_2_Levels of Breast Milk MicroRNAs and Other Non-Coding RNAs Are Impacted by Milk Maturity and Maternal Diet.docx

There is emerging evidence that non-coding RNAs (ncRNAs) within maternal breast milk (MBM) impart unique metabolic and immunologic effects on developing infants. Most studies examining ncRNAs in MBM have focused on microRNAs. It remains unclear whether microRNA levels are related to other ncRNAs, or whether they are impacted by maternal characteristics. This longitudinal cohort study examined 503 MBM samples from 192 mothers to: 1) identify the most abundant ncRNAs in MBM; 2) examine the impact of milk maturity on ncRNAs; and 3) determine whether maternal characteristics affect ncRNAs. MBM was collected at 0, 1, and 4 months post-delivery. High throughput sequencing quantified ncRNAs within the lipid fraction. There were 3069 ncRNAs and 238 microRNAs with consistent MBM presence (≥10 reads in ≥10% samples). Levels of 17 ncRNAs and 11 microRNAs accounted for 80% of the total RNA content. Most abundant microRNAs displayed relationships ([R]>0.2, adj p< 0.05) with abundant ncRNAs. A large proportion of ncRNAs (1269/3069; 41%) and microRNAs (206/238; 86%) were affected by MBM maturity. The majority of microRNAs (111/206; 54%) increased from 0-4 months. Few ncRNAs and microRNAs were affected (adj p < 0.05) by maternal age, race, parity, body mass index, gestational diabetes, or collection time. However, nearly half of abundant microRNAs (4/11) were impacted by diet. To our knowledge this is the largest study of MBM ncRNAs, and the first to demonstrate a relationship between MBM microRNAs and maternal diet. Such knowledge could guide nutritional interventions aimed at optimizing metabolic and immunologic microRNA profiles within MBM.

现有越来越多的研究证据表明，母乳（maternal breast milk, MBM）中的非编码RNA（non-coding RNAs, ncRNAs）可赋予发育中的婴儿独特的代谢与免疫学效应。既往针对母乳中非编码RNA的相关研究多聚焦于微小RNA（microRNAs）。目前仍不清楚微小RNA的表达水平是否与其他非编码RNA存在关联，亦或其表达是否受母体特征的影响。
本项纵向队列研究纳入192名母亲的503份母乳样本，旨在达成三项研究目标：1）鉴定母乳中丰度最高的非编码RNA；2）探究乳汁成熟度对非编码RNA表达的影响；3）明确母体特征是否会对非编码RNA的表达产生作用。研究分别于产后0、1及4个月采集母乳样本，并通过高通量测序（high throughput sequencing）对脂质组分中的非编码RNA进行定量分析。
最终共鉴定出3069种非编码RNA与238种微小RNA，它们在母乳中存在稳定检出情况（在至少10%的样本中，其读段数≥10）。其中17种非编码RNA与11种微小RNA的总表达量占母乳总RNA含量的80%。丰度最高的微小RNA与高丰度非编码RNA之间存在显著相关性（相关系数|R|>0.2，校正后P<0.05）。
大量非编码RNA（1269/3069；41%）与微小RNA（206/238；86%）的表达水平受乳汁成熟度影响。其中多数微小RNA（111/206；54%）的表达量从产后0个月至4个月呈上升趋势。仅少数非编码RNA与微小RNA的表达受母体年龄、种族、产次、体质量指数（body mass index, BMI）、妊娠期糖尿病或样本采集时间的影响（校正后P<0.05）。
然而，近半数丰度较高的微小RNA（4/11）会受母体饮食的影响。
据我们所知，本研究是目前规模最大的母乳非编码RNA相关研究，亦是首次证实母乳微小RNA与母体饮食之间存在关联的研究。此类研究结果可为旨在优化母乳中代谢与免疫学相关微小RNA特征谱的营养干预策略提供理论指导。