巨噬细胞移动抑制因子通过调节长链非编码 RNA MEG3 保护骨髓间充质干细胞免受低氧/缺血诱导的细胞凋亡。
Macrophage migration inhibitory factor protects bone marrow mesenchymal stem cells from hypoxia/ischemia-induced apoptosis by regulating lncRNA MEG3.
机构信息
First Clinical Medicine Institute, Wenzhou Medical University, Wenzhou 325006, China.
Department of Orthopaedics, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325006, China.
出版信息
J Zhejiang Univ Sci B. 2022 Dec 15;23(12):989-1001. doi: 10.1631/jzus.B2200110.
OBJECTIVES
This research was performed to explore the effect of macrophage migration inhibitory factor (MIF) on the apoptosis of bone marrow mesenchymal stem cells (BMSCs) in ischemia and hypoxia environments.
METHODS
The cell viability of BMSCs incubated under hypoxia/ischemia (H/I) conditions with or without pretreatment with MIF or triglycidyl isocyanurate (TGIC) was detected using cell counting kit-8 (CCK-8) analysis. Plasmids containing long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) or β-catenin small interfering RNA (siRNA) were used to overexpress or downregulate the corresponding gene, and the p53 signaling pathway was activated by pretreatment with TGIC. The influences of MIF, overexpression of lncRNA MEG3, activation of the p53 signaling pathway, and silencing of β-catenin on H/I-induced apoptosis of BMSCs were revealed by western blotting, flow cytometry, and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining.
RESULTS
From the results of CCK-8 assay, western blotting, and flow cytometry, pretreatment with MIF significantly decreased the H/I-induced apoptosis of BMSCs. This effect was inhibited when lncRNA MEG3 was overexpressed by plasmids containing MEG3. The p53 signaling pathway was activated by TGIC, and β-catenin was silenced by siRNA. From western blot results, the expression levels of β-catenin in the nucleus and phosphorylated p53 (p-p53) were downregulated and upregulated, respectively, when the lncRNA MEG3 was overexpressed. Through flow cytometry, MIF was also shown to significantly alleviate the increased reactive oxygen species (ROS) level of BMSCs caused by H/I.
CONCLUSIONS
In summary, we conclude that MIF protected BMSCs from H/I-induced apoptosis by downregulating the lncRNA MEG3/p53 signaling pathway, activating the Wnt/β-catenin signaling pathway, and decreasing ROS levels.
目的
本研究旨在探讨巨噬细胞移动抑制因子（MIF）对骨髓间充质干细胞（BMSCs）在缺血缺氧环境中凋亡的影响。
方法
用细胞计数试剂盒-8（CCK-8）分析法检测缺氧/缺血（H/I）条件下用或不用 MIF 或三缩水甘油异氰尿酸酯（TGIC）预处理的 BMSCs 细胞活力。用含有长链非编码 RNA（lncRNA）母源性表达基因 3（MEG3）或β-连环蛋白小干扰 RNA（siRNA）的质粒过表达或下调相应基因，并通过 TGIC 预处理激活 p53 信号通路。通过 Western blot、流式细胞术和末端脱氧核苷酸转移酶（TdT）介导的 dUTP 缺口末端标记（TUNEL）染色揭示 MIF、lncRNA MEG3 的过表达、p53 信号通路的激活以及β-连环蛋白的沉默对 H/I 诱导的 BMSCs 凋亡的影响。
结果
从 CCK-8 测定、Western blot 和流式细胞术的结果来看，MIF 预处理可显著降低 H/I 诱导的 BMSCs 凋亡。用含有 MEG3 的质粒过表达 lncRNA MEG3 可抑制这种作用。TGIC 激活 p53 信号通路，siRNA 沉默β-连环蛋白。从 Western blot 结果可以看出，过表达 lncRNA MEG3 时，核内β-连环蛋白和磷酸化 p53（p-p53）的表达水平分别下调和上调。通过流式细胞术还表明，MIF 还可以显著减轻 H/I 引起的 BMSCs 活性氧（ROS）水平升高。
结论
综上所述，我们得出结论，MIF 通过下调 lncRNA MEG3/p53 信号通路、激活 Wnt/β-连环蛋白信号通路和降低 ROS 水平来保护 BMSCs 免受 H/I 诱导的凋亡。
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