Thirty-two differentially expressed (DE) miRNAs were identified in spleen, including 16 miRNAs between S and C, 13 between R and C, and 13 between S and R. Through integration analysis of DE miRNAs and mRNA, a total of 273 miRNA-target genes were identified. Functional annotation analysis showed that Apoptosis and NOD-like receptor signaling pathway and adaptive immune response were significantly enriched (P < 0.05). Interestingly, apoptosis pathway was significantly enriched in S vs. C, while NOD-like receptor pathway was enriched in R vs. C (P < 0.05). Two miRNAs, gga-miR-101-3p and gga-miR-155, in the hub positions of the miRNA-mRNA regulatory network, were identified as candidates potentially associated with SE infection. These 2 miRNAs directly repressed luciferase reporter gene activity via binding to 3′-untranslated regions of immune-related genes IRF4 and LRRC59; over-expressed gga-miR-155 and interference gga-miR-101-3p in chicken HD11 macrophage cells significantly altered expression of their target genes and decreased the production of pro-inflammatory cytokines.
These findings facilitate better understanding of the mechanisms of host resistance and susceptibility to SE infection in chickens.
Different expression profiles of miRNAs among C, R, and S chickens
(A) Size distribution of sequenced small RNA reads. (B) Venn diagram demonstrates the overlap of differentially expressed (DE) miRNAs among the three groups; numbers are the DE miRNAs in each comparison. (C) Correspondence of miRNAs obtained by high-throughput sequencing and qPCR. C, Controls; R, Resistant; S, Susceptible.