National Institute for Health Research Nottingham Biomedical Research Centre, University of Nottingham, Nottingham, United Kingdom; Nottingham Digestive Diseases Centre, School of Medicine, University of Nottingham, Nottingham, United Kingdom.
Department of Biological Sciences, Clemson University, Clemson, South Carolina, USA.
Gastroenterology. 2021 Jul;161(1):255-270.e4. doi: 10.1053/j.gastro.2021.03.050. Epub 2021 Apr 9.
The molecular mechanisms underlying successful fecal microbiota transplantation (FMT) for recurrent Clostridioides difficile infection (rCDI) remain poorly understood. The primary objective of this study was to characterize alterations in microRNAs (miRs) following FMT for rCDI.
Sera from 2 prospective multicenter randomized controlled trials were analyzed for miRNA levels with the use of the Nanostring nCounter platform and quantitative reverse-transcription (RT) polymerase chain reaction (PCR). In addition, rCDI-FMT and toxin-treated animals and ex vivo human colonoids were used to compare intestinal tissue and circulating miRs. miR inflammatory gene targets in colonic epithelial and peripheral blood mononuclear cells were evaluated by quantitative PCR (qPCR) and 3'UTR reporter assays. Colonic epithelial cells were used for mechanistic, cytoskeleton, cell growth, and apoptosis studies.
miRNA profiling revealed up-regulation of 64 circulating miRs 4 and 12 weeks after FMT compared with screening, of which the top 6 were validated in the discovery cohort by means of RT-qPCR. In a murine model of relapsing-CDI, RT-qPCR analyses of sera and cecal RNA extracts demonstrated suppression of these miRs, an effect reversed by FMT. In mouse colon and human colonoids, C difficile toxin B (TcdB) mediated the suppressive effects of CDI on miRs. CDI dysregulated DROSHA, an effect reversed by FMT. Correlation analyses, qPCR ,and 3'UTR reporter assays revealed that miR-23a, miR-150, miR-26b, and miR-28 target directly the 3'UTRs of IL12B, IL18, FGF21, and TNFRSF9, respectively. miR-23a and miR-150 demonstrated cytoprotective effects against TcdB.
These results provide novel and provocative evidence that modulation of the gut microbiome via FMT induces alterations in circulating and intestinal tissue miRs. These findings contribute to a greater understanding of the molecular mechanisms underlying FMT and identify new potential targets for therapeutic intervention in rCDI.
复发性艰难梭菌感染（rCDI）粪菌移植（FMT）成功的分子机制仍知之甚少。本研究的主要目的是描述 rCDI 行 FMT 后 microRNAs（miRs）的变化。
使用 Nanostring nCounter 平台和定量逆转录（RT）聚合酶链反应（PCR）对来自 2 项前瞻性多中心随机对照试验的血清进行 miRNA 水平分析。此外，rCDI-FMT 和毒素处理的动物和离体人结肠类器官用于比较肠组织和循环 miRs。通过定量 PCR（qPCR）和 3'UTR 报告基因测定评估结肠上皮和外周血单个核细胞中 miR 炎症基因靶标。使用结肠上皮细胞进行机制、细胞骨架、细胞生长和凋亡研究。
miRNA 谱分析显示，与筛选相比，FMT 后 4 周和 12 周时，64 种循环 miRs 上调，其中前 6 种通过 RT-qPCR 在发现队列中得到验证。在复发性-CDI 小鼠模型中，血清和盲肠 RNA 提取物的 RT-qPCR 分析表明，这些 miRs 受到抑制，FMT 可逆转该抑制作用。在小鼠结肠和人结肠类器官中，艰难梭菌毒素 B（TcdB）介导 CDI 对 miRs 的抑制作用。FMT 逆转了 CDI 对 DROSHA 的调节作用。相关性分析、qPCR 和 3'UTR 报告基因测定表明，miR-23a、miR-150、miR-26b 和 miR-28 分别直接靶向 IL12B、IL18、FGF21 和 TNFRSF9 的 3'UTRs。miR-23a 和 miR-150 对 TcdB 表现出细胞保护作用。
这些结果提供了新的、有启发性的证据，表明通过 FMT 调节肠道微生物组会导致循环和肠组织 miRs 的变化。这些发现有助于更好地理解 FMT 的分子机制，并确定 rCDI 治疗干预的新潜在靶点。
