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.

中文翻译：

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弓形虫速殖子腹腔感染小鼠胎盘中 Foxp3 表达的抑制：深入了解 PPARγ/miR-7b-5p/Sp1 信号通路

弓形虫是一种专性细胞内寄生原生动物，感染全球约 30% 的人口。母亲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，并伴有 Forkhead box P3 (Foxp3) 减少。大量研究已证明 microRNA (miRNA) 与弓形虫感染有关，但关于 miRNA 的改变与弓形虫诱导的 Foxp3 下调之间的关系的证据很少。利用定量逆转录聚合酶链反应检测miRNA和Foxp3的转录。采用蛋白印迹法和免疫荧光法检测Foxp3及相关转录因子的表达。通过苏木精和伊红（HE）染色观察小鼠胎盘的结构。为了检查 miR-7b 启动子的活性以及 miR-7b-5p 是否靶向 Sp1 来抑制 Foxp3 表达，我们构建了含有全长/截短/突变 miR-7b 启动子序列或 Sp1 3' 未翻译的野生型/突变体的重组质粒区域（3' UTR）来检测 EL4 细胞中的荧光活性。在弓形虫感染的小鼠中，胎盘中 miR-7b 转录显着升高，而 Foxp3 表达降低。在体外，miR-7b 模拟下调 Foxp3 的表达，而其抑制剂则显着上调 Foxp3 的表达。刺激弓形虫抗原后，miR-7b 启动子活性升高，而共转染缺乏过氧化物酶体增殖物激活受体 γ (PPARγ) 靶位点的突变型 miR-7b 启动子可减轻这种活性。此外，miR-7b 模拟了 Sp1 表达的减少，而 miR-7b 抑制剂则提高了其表达。 miR-7b 模拟物降低了 Sp1 3' 非翻译区 (3' UTR) 的荧光活性，但未能影响共转染缺乏 miR-7b 靶位点的突变体 Sp1 3' UTR 时的荧光活性。弓形虫感染和抗原促进 miR-7b 转录，但抑制 Foxp3 蛋白和基因水平。弓形虫抗原通过 PPARγ 依赖性机制促进 miR-7b 启动子活性。 miR-7b 直接结合 Sp1 3' UTR 以抑制 Sp1 表达。了解弓形虫诱导的 miR-7b 抑制 Foxp3 表达的调节功能可以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。