MicroRNA profiling of the pig periaqueductal grey (PAG) region reveals candidates potentially related to sex-dependent differences

Abstract

Background: MicroRNAs indirectly orchestrate myriads of essential biological processes. A wide diversity of miRNAs of the neurodevelopmental importance characterizes the brain tissue, which, however, exhibits region-specific miRNA profile differences. One of the most conservative regions of the brain is periaqueductal grey (PAG) playing vital roles in significant functions of this organ, also those observed to be sex-influenced. The domestic pig is an important livestock species but is also believed to be an excellent human model. This is of particular importance for neurological research because of the similarity of pig and human brains as well as difficult access to human samples. However, the pig PAG profile has not been characterized so far. Moreover, molecular bases of sex differences connected with brain functioning, including miRNA expression profiles, have not been fully deciphered yet.

Methods: Thus, in this study, we applied next-generation sequencing to characterize pig PAG expressed microRNAs. Furthermore, we performed differential expression analysis between females and males to identify changes of the miRNA profile and reveal candidates underlying sex-related differences.

Results: As a result, known brain-enriched, and new miRNAs which will expand the available profile, were identified. The downstream analysis revealed 38 miRNAs being differentially expressed (DE) between female and male samples. Subsequent pathway analysis showed that they enrich processes vital for neuron growth and functioning, such as long-term depression and axon guidance. Among the identified sex-influenced miRNAs were also those associated with the PAG physiology and diseases related to this region.

Conclusions: The obtained results broaden the knowledge on the porcine PAG miRNAome, along with its dynamism reflected in different isomiR signatures. Moreover, they indicate possible mechanisms associated with sex-influenced differences mediated via miRNAs in the PAG functioning. They also provide candidate miRNAs for further research concerning, i.e., sex-related bases of physiological and pathological processes occurring in the nervous system.

Keywords: Brain; NGS; PAG; Pig; microRNAs.

Fig. 1

The expression pattern Heat Map. The Heat Map depicts the most significant (p value ≤ 0.005) differentially expressed miRNAs and their isomiRs (females vs males; males constitute the reference group) (R Package pheatmap)

Fig. 2

MiRNA interaction network. Global interaction network between the identified differentially expressed miRNAs (females vs. males), and predicted genes as well as long non-coding RNAs, which they target (miRNet web application). Green squares stand for DE microRNAs, red circles denote target genes, while blue circles stand for lncRNAs

Fig. 3

miR-99a-5p interaction network. Interaction network of differentially expressed (females vs. males) miR-99a-5p, and its predicted target genes as well as long non-coding RNAs (miRNet web application). Green squares denote DE microRNAs, red circles stand for target genes, and blue circles stand for lncRNAs

Fig. 4

Prolactin signaling pathway (KEGG ID: hsa04917). A pathway identified as one of the most significantly enriched by the detected differentially expressed miRNAs in females in comparison to males. Green frame denotes genes engaged in the pathway. Genes targeted by identified DE miRNAs are marked yellow (genes included in one pathway) and orange (genes included in more than one pathway). Black arrows denote a molecular interaction or relation, whereas dotted arrows stand for an indirect link or unknown reaction

Fig. 5

Long-term depression (KEGG ID: hsa04730). One of the most significantly enriched pathways by the detected differentially expressed between females and males microRNAs. Green frame stands for genes engaged in the pathway. Genes targeted by detected DE microRNAs are marked yellow (genes included in one pathway) and orange (genes included in more than one pathway). Black arrows stand for a molecular interaction or relation, while dotted arrows stand for an indirect link or unknown reaction

Fig. 6

Axon guidance (KEGG ID: hsa04360). One of the most important pathways, enriched by the identified differentially expressed miRNAs in females vs. males. Green frame stands for genes involved in the pathway. Genes targeted by identified DE microRNAs are marked yellow (genes included in one pathway) and orange (genes included in more than one pathway). Black arrows denote a molecular interaction or relation, while dotted arrows denote an indirect link or unknown reaction

Fig. 7

Heat map of significant GO terms enriched by the DE microRNAs (females vs. males) (DIANA-miRPath v3.0)