Abstract
Objective
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.
概要
目的
探讨巨噬细胞迁移抑制因子(MIF)在缺氧/缺血(H/I)环境下对骨髓间充质干细胞(BMSCs)凋亡的影响。
创新点
(1)阐明了长链非编码RNA(lncRNA)MEG3在H/I诱导的小鼠BMSCs凋亡中的作用;(2)发现MIF可以通过调节lncRNA MEG3/p53信号通路缓解H/I介导的BMSCs凋亡;(3)MIF的细胞保护作用还与激活Wnt/β-连环蛋白(β-catenin)信号通路和降低活性氧(ROS)水平相关。
方法
采用细胞计数试剂8(CCK-8)法检测H/I条件下BMSCs有或无MIF或异氰脲酸甘油三酯(TGIC)预处理对细胞活力的影响。利用含有lncRNA MEG3的质粒或β-catenin小干扰RNA过表达或下调相应基因,并使用TGIC预处理激活p53信号通路,然后进一步检测MIF对H/I环境下BMSCs的保护作用。
结果
CCK-8、蛋白质印迹法和流式细胞术检测结果显示:MIF预处理可显著降低细胞内的ROS水平,改善H/I诱导的BMSCs凋亡;LncRNA MEG3过表达时细胞核内β-catenin的含量明显减少,而p-p53的表达上调;当p53信号通路被激活,β-catenin的表达被抑制时,MIF对H/I环境下BMSCs的保护作用明显减弱。
结论
MIF通过下调lncRNA MEG3/p53信号通路、激活Wnt/β-catenin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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Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 81702132), the Zhejiang Provincial Natural Science Foundation of China (No. LY21H060007), the Projects of Medical and Health Technology Program in Zhejiang Province (No. 2021KY206), and the Wenzhou Public Welfare Science and Technology Research Project (Nos. Y20190267 and Y20210436), China.
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All authors contributed to the study conception and design. Zhibiao BAI and Kai HU performed the experimental research and data analysis, wrote and edited the manuscript. Jiahuan YU and Yizhe SHEN created the figures. Chun CHEN contributed to data analysis and discussion. All authors have read and approved the final manuscript, and therefore, have full access to all data relevant to the study and take responsibility for the integrity and security of such data.
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Zhibiao BAI, Kai HU, Jiahuan YU, Yizhe SHEN, and Chun CHEN declare that they have no conflict of interest.
This study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China (No. wydw2020-0361).
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Bai, Z., Hu, K., Yu, J. et al. Macrophage migration inhibitory factor protects bone marrow mesenchymal stem cells from hypoxia/ischemia-induced apoptosis by regulating lncRNA MEG3. J. Zhejiang Univ. Sci. B 23, 989–1001 (2022). https://doi.org/10.1631/jzus.B2200110
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DOI: https://doi.org/10.1631/jzus.B2200110
Key words
- Macrophage migration inhibitory factor (MIF)
- Long noncoding RNA (lncRNA)
- Maternally expressed gene 3 (MEG3)
- Bone marrow mesenchymal stem cells (BMSCs)
- β-Catenin
- Apoptosis