Department of Gastrointestinal Surgery, Second Affiliated Hospital of Kunming Medical University, No.374, Dianmian Road, Kunming, 650101, Yunnan Province, People's Republic of China.
Mol Neurobiol. 2022 Aug;59(8):5222-5235. doi: 10.1007/s12035-022-02833-3. Epub 2022 Jun 10.
Intestinal ischemia/reperfusion (I/R) injury (IIRI) is associated with high prevalence and mortality rate. Recently, mesenchymal stem cell (MSC) therapy attracted more attentions. However, the function and regulatory mechanism of MSC-derived exosomal miRNAs during IIRI remain largely uninvestigated. The in vitro and in vivo IIRI models were established. MSC were characterized by immunofluorescent staining and flow cytometry. Purified exosomes were characterized by transmission electron microscopy (TEM), flow cytometry, and western blot. The expression of key molecules was detected by western blot and qRT-PCR. CCK-8, TUNEL, and transepithelial electrical resistance (TER) assays were employed to assess cell viability, apoptosis, and intestinal integrity, respectively. Pre-miR-34A m modification was evaluated by methylated RNA immunoprecipitation (MeRIP)-qPCR. RNA pull-down and RIP were used to validate the direct association between pre-miR-34A and IGF2BP3. MSC-derived exosomal miR-34a-5p alleviated OGD/R-induced injury. In addition, MSC ameliorated OGD/R-induced injury through METTL3 pathway. Mechanistic study revealed that miR-34a-5p was modulated by METTL3/IGF2BP3-mediated mA modification in MSC. The in vitro and in vivo functional experiments revealed that MSC secreted exosomal miR-34a-5p and ameliorated IIRI through METTL3/IGF2BP3-mediated mA modification of pre-miR-34A. MSC promoted the secretion of exosomal miR-34a-5p and improved intestinal barrier function through METTL3/IGF2BP3-mediated pre-miR-34A mA modification.
肠缺血/再灌注（I/R）损伤（IIRI）与高患病率和高死亡率相关。最近，间充质干细胞（MSC）治疗引起了更多关注。然而，MSC 衍生的外泌体 miRNA 在 IIRI 中的功能和调节机制在很大程度上仍未得到研究。建立了体外和体内 IIRI 模型。通过免疫荧光染色和流式细胞术对 MSC 进行了表征。通过透射电子显微镜（TEM）、流式细胞术和 Western blot 对纯化的外泌体进行了表征。通过 Western blot 和 qRT-PCR 检测了关键分子的表达。CCK-8、TUNEL 和跨上皮电阻（TER）测定分别用于评估细胞活力、细胞凋亡和肠完整性。通过甲基化 RNA 免疫沉淀（MeRIP）-qPCR 评估 pre-miR-34A m 修饰。使用 RNA 下拉和 RIP 验证 pre-miR-34A 与 IGF2BP3 之间的直接关联。MSC 衍生的外泌体 miR-34a-5p 减轻了 OGD/R 诱导的损伤。此外，MSC 通过 METTL3 途径改善了 OGD/R 诱导的损伤。机制研究表明，miR-34a-5p 受 MSC 中 METTL3/IGF2BP3 介导的 mA 修饰调节。体外和体内功能实验表明，MSC 分泌外泌体 miR-34a-5p，并通过 METTL3/IGF2BP3 介导的 pre-miR-34A mA 修饰改善 IIRI。MSC 通过 METTL3/IGF2BP3 介导的 pre-miR-34A mA 修饰促进外泌体 miR-34a-5p 的分泌并改善肠屏障功能。
