Department of Pathogen Biology, School of Medicine, Nantong University, 19 Qixiu Road, Nantong, 226001, Jiangsu, People's Republic of China.
Department of Obstetrics and Gynecology, Affiliated Hospital of Nantong University, Nantong, 226001, Jiangsu, People's Republic of China.
Parasit Vectors. 2024 Apr 17;17(1):189. doi: 10.1186/s13071-024-06262-0.
Toxoplasma gondii, an obligate intracellular parasitic protozoa, infects approximately 30% of the global population. Contracting T. gondii at the primary infection of the mother can result in neonatal microcephaly, chorioretinitis, hydrocephalus, or mortality. Our previous study indicated that pregnant mice infected with T. gondii displayed a decrease in both the number and the suppressive ability of regulatory T cells, accompanied by the reduced Forkhead box P3 (Foxp3). Numerous studies have proved that microRNAs (miRNAs) are implicated in T. gondii infection, but there is meager evidence on the relationship between alterations of miRNAs and downregulation of Foxp3 induced by T. gondii.
Quantitative reverse transcription polymerase chain reaction was utilized to detect the transcriptions of miRNAs and Foxp3. Protein blotting and immunofluorescence were used to detect the expressions of Foxp3 and related transcription factors. The structure of mouse placenta was observed by hematoxylin and eosin (HE) staining. To examine the activity of miR-7b promoter and whether miR-7b-5p targets Sp1 to suppress Foxp3 expression, we constructed recombinant plasmids containing the full-length/truncated/mutant miR-7b promoter sequence or wildtype/mutant of Sp1 3' untranslated region (3' UTR) to detect the fluorescence activity in EL4 cells.
In T. gondii-infected mice, miR-7b transcription was significantly elevated, while Foxp3 expression was decreased in the placenta. In vitro, miR-7b mimics downregulated Foxp3 expression, whereas its inhibitors significantly upregulated Foxp3 expression. miR-7b promoter activity was elevated upon the stimulation of T. gondii antigens, which was mitigated by co-transfection of mutant miR-7b promoter lacking peroxisome proliferator-activated receptor γ (PPARγ) target sites. Additionally, miR-7b mimics diminished Sp1 expression, while miR-7b inhibitors elevated its expression. miR-7b mimics deceased the fluorescence activity of Sp1 3' untranslated region (3' UTR), but it failed to impact the fluorescence activity upon the co-transfection of mutant Sp1 3' UTR lacking miR-7b target site.
T. gondii infection and antigens promote miR-7b transcription but inhibit Foxp3 protein and gene levels. T. gondii antigens promote miR-7b promoter activity by a PPARγ-dependent mechanism. miR-7b directly binds to Sp1 3' UTR to repress Sp1 expression. Understanding the regulatory functions by which T. gondii-induced miR-7b suppresses Foxp3 expression can provide new perspectives for the possible therapeutic avenue of T. gondii-induced adverse pregnancy outcomes.
刚地弓形虫是一种专性细胞内寄生的原生动物，全球约有 30%的人口受到感染。母体初次感染弓形虫可导致新生儿小头畸形、脉络膜视网膜炎、脑积水或死亡。我们之前的研究表明，感染弓形虫的怀孕小鼠的调节性 T 细胞数量和抑制能力均下降，叉头框 P3（Foxp3）减少。大量研究证明 microRNAs（miRNAs）参与了弓形虫感染，但关于 miRNAs 的改变与弓形虫诱导的 Foxp3 下调之间的关系，证据很少。
采用定量逆转录聚合酶链反应检测 miRNAs 和 Foxp3 的转录水平。蛋白印迹和免疫荧光检测 Foxp3 和相关转录因子的表达。苏木精和伊红（HE）染色观察小鼠胎盘的结构。为了检测 miR-7b 启动子的活性以及 miR-7b-5p 是否通过靶向 Sp1 抑制 Foxp3 的表达，我们构建了包含全长/截短/突变 miR-7b 启动子序列或野生型/突变型 Sp1 3'非翻译区（3'UTR）的重组质粒，以检测 EL4 细胞中的荧光活性。
在弓形虫感染的小鼠中，miR-7b 的转录明显升高，而胎盘内 Foxp3 的表达降低。在体外，miR-7b 模拟物下调 Foxp3 的表达，而其抑制剂则显著上调 Foxp3 的表达。弓形虫抗原刺激可提高 miR-7b 启动子活性，而过氧化物酶体增殖物激活受体γ（PPARγ）靶位点缺失的突变 miR-7b 启动子可减轻该活性。此外，miR-7b 模拟物降低 Sp1 的表达，而 miR-7b 抑制剂则增加其表达。miR-7b 模拟物降低 Sp1 3'UTR 的荧光活性，但在共转染缺乏 miR-7b 靶位点的突变 Sp1 3'UTR 时，其荧光活性并未受到影响。
弓形虫感染和抗原促进 miR-7b 的转录，但抑制 Foxp3 蛋白和基因水平。弓形虫抗原通过 PPARγ 依赖的机制促进 miR-7b 启动子活性。miR-7b 直接结合 Sp1 3'UTR 抑制 Sp1 的表达。了解弓形虫诱导的 miR-7b 抑制 Foxp3 表达的调节功能，可为弓形虫诱导的不良妊娠结局的可能治疗途径提供新的视角。
