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miR-509-5p anti-infection response for mycoplasma pneumonia in sheep by targeting NF-κB pathway
Mengting Zhu1, Shaoqi Cao2, Wei Zheng3
1College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang, China; State Key Laboratory of Sheep Genetic Improvement and Healthy Production, Xinjiang Academy of Agricultural and Reclamation Science, Shihezi, China.
Abstract:
MicroRNAs play a key role in Mannan-binding lectin-mediated resistance to Mycoplasma ovipneumoniae pneumonia, by regulating the translation of mRNAs of target genes, thereby regulating the immune response. Additionally, TRAF6 is a key molecule in Toll-like receptor signal transduction, which mediates inflammation and apoptosis signaling pathways and is widely involved in inflammation and immune response. While the molecular regulation mechanism has not been reported. In this study, we screened differentially expressed miRNAs and genes of Anti-infection for M. pneumonia on Sheep, through relevant bioinformatics analysis. Further, the effect of differential expression of NF-κB signaling pathway related genes on the molecular mechanism of M. pneumonia was detected. We used miRNA-mRNA integrated analysed, the target gene TRAF6 of miR-509-5p was selected. TRAF6 dual luciferase reporter vector was co-transfected into HEK 293T cells and primary sheep respiratory mucosal epithelial cells to detect changes in luciferase activity. qRT-PCR was used to analyze the effect of miR-509-5p on the expression and regulation of TRAF6 and other genes related to the NF-κB signaling pathway. The result confirmed that TRAF6 was a target gene of miR-509-5p. Compared with miR-509-5p-NC group, the luciferase activity of miR-509-5p group was significantly down-regulated (P < 0.01). Further, in sheep respiratory mucosal epithelial cells, miR-509-5p mimic could significantly down-regulate the fold change value of TRAF6 (P < 0.01). On the contrary, miR-509-5p-inhibitor up-regulated the fold change value of TRAF6 (P < 0.05). Interestingly, the expression levels of other genes were different. Among them, miR-509-5p mimic significantly up-regulated TLR4 and IRAK4 (P < 0.05), significantly down-regulated TAK1 (P < 0.05) and NF-κB (P < 0.01). miR-509-5p-inhibitor significantly up-regulated NF-κB (P < 0.05) and TAK1 (P < 0.01). miR-509-5p targets TRAF6 to affect the expression of downstream genes, which negatively regulates the NF-κB pathway, thereby affecting the inflammatory response.
Insights
This study reveals that miR-509-5p targets TRAF6, negatively regulating the NF-κB pathway and sheep
Area of Science:
- Veterinary immunology
- Molecular biology
- Microbial pathogenesis
Background:
- MicroRNAs regulate immune responses and Mannan-binding lectin-mediated resistance to Mycoplasma ovipneumoniae.
- TRAF6 is crucial in Toll-like receptor signaling, inflammation, and apoptosis.
- The molecular mechanisms of M. ovipneumoniae infection in sheep remain unclear.
Purpose of the Study:
- To investigate the role of microRNAs and NF-κB signaling in sheep's anti-infection response to M. ovipneumoniae.
- To identify miRNA-mRNA interactions regulating M. ovipneumoniae infection.
- To elucidate the regulatory mechanism of miR-509-5p on TRAF6 and the NF-κB pathway.
Main Methods:
- Bioinformatic analysis of differentially expressed miRNAs and genes in sheep infected with M. ovipneumoniae.
- miRNA-mRNA integrated analysis to identify target genes.
- Luciferase reporter assays in HEK 293T and primary sheep respiratory mucosal epithelial cells.
- Quantitative reverse transcription PCR (qRT-PCR) to assess gene expression.
Main Results:
- TRAF6 was identified as a direct target gene of miR-509-5p.
- miR-509-5p significantly down-regulated TRAF6 expression in sheep respiratory mucosal epithelial cells.
- miR-509-5p mimic modulated the expression of NF-κB pathway genes, including TLR4, IRAK4, TAK1, and NF-κB.
- miR-509-5p negatively regulates the NF-κB pathway by targeting TRAF6.
Conclusions:
- miR-509-5p plays a critical role in the immune response to M. ovipneumoniae in sheep.
- The miR-509-5p/TRAF6 axis is a key regulator of the NF-κB signaling pathway.
- Targeting this pathway could offer therapeutic strategies for Mycoplasma pneumonia in sheep.
