Deep RNA sequencing reveals that microRNAs play a key role in lactation in rats

Understanding the regulatory contribution of maternal physiology to difficulties with lactation is beneficial to both mother and infant. MicroRNAs (miRNAs), a type of noncoding RNA, may be involved in the regulation of mammary gland development and function. In the present study, a deep RNA sequencing (RNA-seq) technique was used to compare the expression profile of miRNAs and mRNAs of 2 pooled RNA samples from day 1 and day 7 postpartum (n = 1/d) rat (Rattus norvegicus) mammary glands to identify key miRNAs and their target genes that may control the rate-limiting steps of lactation. A total of 395 and 400 known miRNAs were identified in days 1 and 7 postpartum rat mammary samples, respectively. Compared with day 1 postpartum, 27 miRNAs were differentially expressed at day 7 postpartum. The expression differences between lactation periods were further analyzed by real-time quantitative polymerase chain reaction (qPCR) (n = 5). The ΔΔCt values of rno-miR-30, rno-miR-1, rno-miR-145-3p, rno-miR-142, rno-miR-7a-5p, rno-miR-3571, rno-miR-224-5p, rno-miR-362-5p, rno-miR-342-3p, rno-miR-322-3p, rno-miR-18a-5p, and rno-miR-202-5p between the 2 libraries varied from 0.64 to 9.44; the ΔΔCt values of rno-miR-133, rno-miR-190a-5p, rno-miR-27a-5p, rno-miR-451-5p, rno-miR-3120, rno-miR-23a-5p, rno-miR-20a-3p, rno-miR-92a-1-5p, and rno-miR-134-5p between the 2 libraries varied from -1.02 to -4.37 (P < 0.05). The intersection of the expressed mRNA genes from RNA-seq and putative target genes of differentially expressed miRNAs, termed mammary gland target genes (MTGs), was analyzed. The results indicated that 1259 MTGs overlapped between the 2 gene sets. The expression of 14 randomly selected genes of the MTGs was further confirmed by real-time qPCR (R(2) = 0.86, P < 0.01). The downregulated MTGs were enriched for the pathways involved in lipid biosynthesis. This gene cluster included 24 lipid metabolic process-related genes, which were putative targets of 10 differentially expressed miRNAs. These results will be helpful in discovering the biologic underpinnings of poor lactation performance in women attempting to breastfeed.