BACKGROUND Toxoplasma gondii is an apicomplexan protozoan parasite that can cause serious clinical illnesses in both humans and animals. microRNAs (miRNAs) are non-protein-coding RNAs that can regulate the expression of target genes. A previous study found that many miRNAs were differentially expressed after T. gondii infection and exert significant effects and revealed that both host survival and the virulence of different strains can be regulated by different miRNAs. Macrophages play an important role in T. gondii infection, but few studies have investigated the relationship between miRNAs and porcine alveolar macrophages infected with T. gondii. METHODS Porcine alveolar macrophages (3D4-21) were infected with the RH (Type I) and Me49 (Type II) strains of T. gondii for 12 h and 24 h and then harvested. miRNA libraries were generated using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA), and the miRNA expression levels were estimated based on transcripts per million reads (TPM). RESULTS Our study generated six miRNA expression profiles from macrophages infected with RH and Me49 compared with the control groups. The comparison of the T. gondii-infected and uninfected samples identified 81 differentially expressed miRNAs, including 36 novel miRNAs and 45 mature miRNAs. The target genes of these differentially expressed miRNAs were predicted using miRanda software, and ssc-miR-127 and ssc-miR-143-3p were predicted to regulate nitric oxide synthase 1 (NOS1) and nitric oxide synthase 3 (NOS3), respectively, which play essential roles in synthesizing nitric oxide (NO) by oxidizing L-arginine. These genes were differentially expressed in both the RH- and Me49-infected groups. A KEGG enrichment analysis indicated that the predicted target genes were involved in multiple signaling pathways, including FcγR-mediated phagocytosis, the AMPK signaling pathway, the mTOR signaling pathway, and the FcγRI signaling pathway, all of which are indispensable for the normal functioning of porcine alveolar macrophages. CONCLUSIONS Our results provide data on the miRNA profile of porcine alveolar macrophages infected with T. gondii. To our knowledge, this study provides the first demonstration of the relationship between miRNA and macrophages of swine origin. Understanding the functions of these regulated miRNAs will aid the investigation of T. gondii infectious diseases, and the differentially expressed miRNAs might be candidate drug targets for T. gondii infection in pigs.

中文翻译：

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弓形虫感染后猪肺泡巨噬细胞中microRNA的表达谱。

背景技术弓形虫是一种apicomplexan的原生动物寄生虫，可在人类和动物中引起严重的临床疾病。微小RNA（miRNA）是非蛋白质编码的RNA，可以调节靶基因的表达。先前的研究发现，弓形虫感染后许多miRNA差异表达并发挥显著作用，并表明宿主存活和不同菌株的毒力都可以通过不同的miRNA来调节。巨噬细胞在弓形虫感染中起重要作用，但很少有研究调查miRNA与感染弓形虫的猪肺泡巨噬细胞之间的关系。方法分别用弓形虫的RH（I型）和Me49（II型）菌株感染猪肺泡巨噬细胞（3D4-21）12 h和24 h，然后收获。使用用于Illumina®的NEBNext®多重小RNA文库制备套件（美国NEB）生成miRNA文库，并基于每百万读取的转录本（TPM）估算miRNA表达水平。结果与对照组相比，我们的研究从感染RH和Me49的巨噬细胞中产生了6个miRNA表达谱。弓形虫感染和未感染样品的比较确定了81种差异表达的miRNA，包括36种新颖的miRNA和45种成熟的miRNA。使用miRanda软件预测这些差异表达的miRNA的靶基因，并预测ssc-miR-127和ssc-miR-143-3p分别调节一氧化氮合酶1（NOS1）和一氧化氮合酶3（NOS3），它们通过氧化L-精氨酸在合成一氧化氮（NO）中起重要作用。这些基因在RH和Me49感染组中差异表达。KEGG富集分析表明，预测的靶基因参与了多种信号通路，包括FcγR介导的吞噬作用，AMPK信号通路，mTOR信号通路和FcγRI信号通路，所有这些对于猪的正常功能都是必不可少的。肺泡巨噬细胞。结论我们的结果提供了感染弓形虫的猪肺泡巨噬细胞的miRNA谱数据。就我们所知，这项研究首次证明了miRNA与猪源巨噬细胞之间的关系。了解这些受调控的miRNA的功能将有助于弓形虫感染性疾病的研究，并且差异表达的miRNA可能是T的候选药物靶标。