Identification of Differentially Expressed Micro RNAs in Peripheral Blood Mononuclear Cells of Topsoil-Exposed Piglets

Exposure of immature immune systems to non-hygienic conditions has been shown to improve future immune function in humans and growth performance in swine. Long-term effects of exposure to a “dirty” environment indicate modulation in physiological programming or epigenetic regulation related to life-long immunity as opposed to short-term transcriptional effects. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that modulate whole pathways in response to the environment that may permanently alter transcription. In our previous studies, exposure to topsoil prior to weaning improved post-weaning growth performance. Therefore, we wanted to determine if exposure to topsoil would alter miRNA abundance. Piglets were selected from litters (PIC 29 x 380) that farrowed within 24 h of each other, and randomly assigned to rearing environments without (C; n=8) or with (S; n=5) topsoil from d 4 to 21 of lactation. Piglets were weaned and transferred to a nursery facility at d 21. Peripheral blood mononuclear cells (PBMC) were collected from piglets at 11, 20, and 56 d of age. Small RNA (18-30 nt) libraries generated from high quality PBMC-derived total RNA (RQI= 7.4-9.9) for 39 samples were sequenced using Illumina^® technology. Sequenced reads were quality-checked using FastQC and then used for novel miRNA identification or differential expression analysis. Reads were aligned by TopHat/Bowtie2 to Sscrofa 10.2 reference genome (Ensembl v84) and binned by gene annotation with HTSeq. A total of 583 miRNAs were identified using mirDeep2, among which 413 constituted novel miRNAs (false discovery rate, FDR < 0.15). Annotated miRNAs (n=170; Ensembl v84) were analyzed for differential expression following 75^th quantile normalization. The miRNA expression was modeled in PROC Glimmix of SAS where treatment, piglet age, and interaction of treatment and age were fit as fixed effects; piglet was fit as a random effect; and piglet gender, sow parity, litter size, and RQI were fit as covariates. To control the FDR, q-values were calculated for all miRNAs in R. Twenty-three known miRNAs were identified as differentially expressed across the topsoil treatment by piglet age interaction (q < 0.1). These miRNAs include miR-29, which regulates innate and adaptive immune responses to intracellular bacterial infection; miR-31, which regulates gut development and homeostasis; and other miRNAs involved in tumor suppression, apoptosis, T-cell function, lipid metabolism, and growth and lipid deposition. Additional research is needed to investigate the potential that these miRNA reprogram the epigenome of young piglets to enhance growth performance beyond the period of environmental exposure.