Rong Hui, Jiao Hanwei, Hao Yongchang, Pang Feng, Li Guohua, Peng Dongmei, Li Yaying, Wang Yuanzhi, Zhang Hui, Fan Quanshui, Wang Fengyang, Chen Chuangfu, Du Li
1 College of Agriculture, Hainan University, Hainan Key Laboratory of Tropical Animal Reproduction and Breeding and Epidemic Disease Research, Animal Genetic Engineering Key Laboratory of Haikou, Haidian Island, Haikou, People's Republic of China.
2 College of Animal Science and Technology, Shihezi University, Shihezi, People's Republic of China.
Innate Immun. 2017 Jul;23(5):424-431. doi: 10.1177/1753425917707025. Epub 2017 Apr 26.
Innate recognition of Brucella spp. is a key step in the activation of inflammation. CD14 binds PAMPs and is involved in LPS-induced pro-inflammatory cytokine release. Previously we showed that knock down of CD14 in RAW264.7 macrophages disrupted Brucella-host interactions. However, its effect on the macrophage microRNA (miRNA) expression profile, especially after stimulation by Brucella infection, is still unclear. To identify miRNAs involved in the macrophage response to Brucella infection, we performed miRNA expression profiling of CD14 knock-down RAW264.7 (224.3) macrophages infected with Brucella melitensis, and demonstrated, for the first time, that CD14 knock down significantly up-regulated the expression of mmu-miR-199a-3p and mmu-miR-183-5p in these conditions. These miRNAs have a well-characterized association with the target genes involved in immune response, inflammatory response, innate immune response, apoptosis processes, anti-apoptosis, cytokine production and cytokine-mediated signaling pathways. Among the 104 inflammation-related candidate target genes of mmu-miR-199a-3p and mmu-miR-183-5p in the 224.3 B. melitensis group cells, the expression of the Cbl-b, a potential target of mmu-miR-199a-3p, was confirmed to be down-regulated using qRT-PCR and Western blot analysis. Our findings suggest that CD14 functions in the Brucella-host interaction may be through altered miRNA expression, and regulation of Cbl-b proteins.
布鲁氏菌属的天然识别是炎症激活的关键步骤。CD14结合病原体相关分子模式并参与脂多糖诱导的促炎细胞因子释放。此前我们发现，在RAW264.7巨噬细胞中敲低CD14会破坏布鲁氏菌与宿主的相互作用。然而，其对巨噬细胞微小RNA（miRNA）表达谱的影响，尤其是在布鲁氏菌感染刺激后的影响，仍不清楚。为了鉴定参与巨噬细胞对布鲁氏菌感染反应的miRNA，我们对感染了羊种布鲁氏菌的CD14敲低的RAW264.7（224.3）巨噬细胞进行了miRNA表达谱分析，并首次证明在这些条件下敲低CD14会显著上调mmu-miR-199a-3p和mmu-miR-183-5p的表达。这些miRNA与参与免疫反应﹑炎症反应﹑天然免疫反应﹑凋亡过程﹑抗凋亡﹑细胞因子产生和细胞因子介导的信号通路的靶基因有明确的关联。在224.3羊种布鲁氏菌组细胞中mmu-miR-199a-3p和mmu-miR-183-5p的104个炎症相关候选靶基因中，使用qRT-PCR和蛋白质印迹分析证实mmu-miR-199a-3p的潜在靶标Cbl-b的表达下调。我们的研究结果表明，CD14在布鲁氏菌与宿主相互作用中的功能可能是通过改变miRNA表达和调节Cbl-b蛋白来实现的。
