Abstract: Objective To evaluate the differential miRNA expression of breast milk exosome in premature and full-term groups, and to analyze the regulatory pathways by bioinformatics, so as to provide guidance and scientific basis for the growth and development of premature infants and the prevention and treatment of related diseases. Methods From August 2020 to June 2021, breast milk samples from 13 premature(premate group)and 9 full-term infants(full-term group)in the Department of Child Health Care of the Second Affiliated Hospital of Nanjing Medical University were collected to extract exosomes. The miRNAs of two groups of breast milk exosomes were sequenced by high-throughput sequencing. According to the sequencing results, miRNA expression profiles of milk exosome were analyzed. Biological function software was used to carry out GO and KEGG pathway analysis of differential miRNA. Results The expression of miRNA in human milk exosomes was rich, especially hsa-miR-148a-3p,hsa-let-7b-5p, hsa-let-7g-5p, hsa-miR-22-3p, hsa-miR-99a-5p, hsa-miR-200, hsa-miR-146b-5p and hsa-miR-26a-5p were relatively high expressed in preterm group and full-term group. Differential expression analysis showed that compared with full-term infant breast milk, 7 miRNAs were up-regulated(log₂|fold change|=2.803, 2.714, 1.632, 2.360, 1.350, 3.387, 2.137, respectively), and 5 miRNAs were down-regulated(log₂|fold change|＝-2.553,-2.197,-2.771,-1.395,-1.136, respectively)(|fold change>2|, P<0.05) in breast milk for preterm infants. In these differential expressed miRNAs, down-regulated miR-29b(P=0.001) and up-regulated miR-133a-3p(P=0.004) were associated with inflammation, and up-regulated miR-126-5p(P=0.021) and miR-126-3p(P=0.041) were associated with lipid metabolism. The fatty acid biosynthesis pathway was obviously enriched in preterm group. MiR-7-5p, miR-29b-3p and miR-100-5p played a role in the fatty acid synthesis pathway. Conclusions Exosomal miRNAs are rich in breast milk, and have significant differences between preterm and full-term infants' mothers. The differentially expressed miRNA in preterm infants treast milk may be related to inflammation and promote the growth and development of preterm infants through the fatty acid biosynthesis pathway.

Key words: breast milk, miRNA, preterm infants, exosome

摘要： 目的 初步评估母乳外泌体miRNA在早产儿和足月儿母乳中的差异表达,利用生物信息学分析调控途径,为早产儿生长发育和相关疾病防治提供指导和科学依据。方法 2020年8月—2021年6月在南京医科大学第二附属医院儿童保健科收集13例早产母乳喂养儿(早产组)和9例足月母乳喂养儿(足月组)的母乳样本,提取母乳中的外泌体,对外泌体miRNA进行高通量测序,分析两组母乳外泌体miRNA表达谱;筛选早产组和足月组母乳差异表达的外泌体miRNA;用生物学功能软件进行GO及KEGG分析。结果 母乳外泌体miRNA表达丰富,hsa-let-7b-5p、hsa-let-7g-5p、hsa-miR-148a-3p、hsa-miR-22-3p、hsa-miR-99a-5p、hsa-miR-200、hsa-miR-146b-5p、hsa-miR-26a-5p等miRNA在早产组和足月组母乳中均相对高表达。与足月组母乳相比,早产组母乳有7个上调(差异倍数对数值分别是2.803、2.714、1.632、2.360、1.350、3.387、2.137),5个下调(差异倍数对数值分别是-2.553、-2.197、-2.771、-1.395、-1.136,P＜0.05),其中差异表达的miRNA中早产儿母乳组下调的miR-29b(P=0.001)、上调的miR-133a-3p(P=0.004)与炎症相关,上调的miR-126-5p(P=0.021)、miR-126-3p(P=0.041)与脂质代谢相关。KEGG通路分析显示脂肪酸生物合成通路显著富集,miR-7-5p、miR-29b-3p和miR-100-5p在脂肪酸合成通路发挥作用。结论 早产儿与足月儿母亲的母乳外泌体miRNA含量丰富且存在显著差异,早产儿母亲母乳中差异表达的miRNA可能与炎症相关,并通过脂肪酸生物合成通路促进早产儿的生长和发育。

关键词: 母乳, 微小核糖核酸, 早产儿, 外泌体