Integration of lncRNA and mRNA Transcriptome Analyses Reveals Genes and Pathways Potentially Involved in Calf Intestinal Growth and Development during the Early Weeks of Life

Abstract

A better understanding of the factors that regulate growth and immune response of the gastrointestinal tract (GIT) of calves will promote informed management practices in calf rearing. This study aimed to explore genomics (messenger RNA (mRNA)) and epigenomics (long non-coding RNA (lncRNA)) mechanisms regulating the development of the rumen and ileum in calves. Thirty-two calves (≈5-days-old) were reared for 96 days following standard procedures. Sixteen calves were humanely euthanized on experiment day 33 (D33) (pre-weaning) and another 16 on D96 (post-weaning) for collection of ileum and rumen tissues. RNA from tissues was subjected to next generation sequencing and 3310 and 4217 mRNAs were differentially expressed (DE) between D33 and D96 in ileum and rumen tissues, respectively. Gene ontology and pathways enrichment of DE genes confirmed their roles in developmental processes, immunity and lipid metabolism. A total of 1568 (63 known and 1505 novel) and 4243 (88 known and 4155 novel) lncRNAs were detected in ileum and rumen tissues, respectively. Cis target gene analysis identified BMPR1A, an important gene for a GIT disease (juvenile polyposis syndrome) in humans, as a candidate cis target gene for lncRNAs in both tissues. LncRNA cis target gene enrichment suggested that lncRNAs might regulate growth and development in both tissues as well as posttranscriptional gene silencing by RNA or microRNA processing in rumen, or disease resistance mechanisms in ileum. This study provides a catalog of bovine lncRNAs and set a baseline for exploring their functions in calf GIT development.

Keywords: calf; cis target gene; gastrointestinal tract; ileum; lncRNA; mRNA; pathways; rumen.

Figure 1

Differentially expressed genes and their enriched gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in rumen tissues. (a) Differentially expressed messenger RNAs (mRNAs); (b) top GO terms and (c) KEGG pathways. ECM: Extracellular matrix.

Figure 2

Differentially expressed genes and their enriched GO terms and KEGG pathways in ileum tissues. (a) Differentially expressed mRNAs; (b) top GO terms and (c) KEGG pathways. MAPK: Mitogen-activated protein kinase; mTOR: Mechanistic target of rapamycin.

Figure 3

Gene ontology terms enriched for rumen long non-coding RNA (lncRNA) cis target genes. Only the relevant or highly significantly enriched terms are shown. See Supplementary Table S9a for detailed results. GTPase: Single GTPase (guanosine triphosphate); NADH: Nicotinamide adenine dinucleotide reduced form.

Figure 4

Kyoto Enciclopedia of Genes and Genomes pathways enriched for rumen lncRNA potential cis target genes. ErbB: Epidermal growth factor receptor; Fc: Fragment crystallisable region; NF-κB: Nuclear factor-κB.

Figure 5

Gene ontology terms enriched for ileum lncRNA potential cis target genes.

Figure 6

Kyoto Enciclopedia of Genes and Genomes pathways enriched for ileum lncRNA potential cis target genes. TRP: Transient receptor potential.

Figure 7

LncRNAs and their potential cis target genes in (a) rumen and (b) ileum tissues.

Figure 8

Quantitative real time PCR (qPCR) validation of the RNA-Sequencing (RNA-Seq) expression results of (a) mRNAs (five differentially expressed (DE) and three non-DE or potential housekeeping mRNAs) in ileum tissue and (b) mRNAs (five differentially expressed and two non-DE) and two lncRNAs in rumen tissue.