The Mediation of miR-34a/miR-449c for Immune Cytokines in Acute Cold/Heat-Stressed Broiler Chicken

SIMPLE SUMMARY: In the intensive and scale poultry industry, the level of heat stress (HS) directly affects the growth, development, and production performance of poultry. To alleviate the adverse effects of stress in broilers, microRNA (miRNA) was regarded as a potential regulator of immune cytokines. In this study, through the sequencing analysis of spleens after cold/heat stress, we found that 33 and 37 miRNA were differentially expressed in the heat stress group compared with the normal (NS) group and cold stress (CS) group, respectively. The differential miRNA were mainly involved in biological processes such as the cytokine–cytokine receptor interaction. To further understand the miRNA-mediated effect of heat stress on the immune level of chickens, we selected miR-34a and miR-449c as the research objects, predicted and verified that interleukin 2 (IL-2) and interleukin 12α (IL-12α) were the target genes of miR-34a and miR-449c. Coupled with the analysis of the expression of other cytokines, we found that miRNA could change the expression of immune cytokines directly or indirectly. This discovery provides a new insight into the mediation of miRNA for immune cytokines in acute cold/heat stressed broiler chicken. ABSTRACT: An increasing amount of evidence has revealed that microRNAs (miRNAs) participated in immune regulation and reaction to acute cold and heat stresses. As a new type of post-transcriptional regulatory factor, miRNA has received widespread attention; However, the specific mechanism used for this regulation still needs to be determined. In this study, thirty broilers at the same growth period were divided into three groups and treated with different temperature and humidity of CS (10–15 °C and 90% Relative Humidity (RH)), HS (39 °C and 90% RH), and NS (26 °C and 50–60% RH) respectively. After 6 h, splenic tissues were collected from all study groups. miRNA sequencing was performed to identify the differentially expressed miRNAs (DEMs) between HS, CS, and NS. We found 33, 37, and 7 DEMs in the HS-NS, HS-CS, CS-NS group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that DEMs were significantly enriched in cytokine–cytokine receptor interaction and functioned as the cellular responders to stress. We chose two miRNA, miR-34a and miR-449c, from the same family and differential expressed in HS-CS and HS-NS group, as the research objects to predict and verify the target genes. The dual-luciferase reporter assay and quantitative real-time PCR (qRT-PCR) confirmed that two cytokines, IL-2 and IL-12α, were the direct target genes of miR-34a and miR-449c. To further understand the mediation mechanism of miRNAs in acute cold/heat-stressed broiler chicken, a splenic cytokines profile was constructed. The results showed that IL-1β was strongly related to acute heat stress in broiler chicken, and from this we predicted that the increased expression of IL-1β might promote the expression of miR-34a, inducing the upregulation of interferon-γ (INF-γ) and IL-17. Our finds have laid a theoretical foundation for the breeding of poultry resistance and alleviation of the adverse effects of stress.