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Bone Marrow Mesenchymal Stem Cell-Derived Exosomal KLF4 Alleviated Ischemic Stroke Through Inhibiting N6-Methyladenosine Modification Level of Drp1 by Targeting lncRNA-ZFAS1

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Abstract

Ischemic stroke has become a serious public health problem that causes high rates of death and disability. Bone marrow mesenchymal stem cell (BMSC)-derived exosomes have shown promising therapeutic results in IS, while the underlying mechanisms need further investigation. Cell and mice models were established through oxygen–glucose deprivation/reoxygenation (OGD/R) treatment and middle cerebral artery occlusion (MCAO)/reperfusion. Exosomes were isolated from BMSCs. Related gene and protein expression was measured by qRT-PCR, Western blotting, and immunofluorescence analysis. The biological functions of treated cells and tissues were analyzed by MTT, ELISA, JC-1, flow cytometry, TTC staining, or TUNEL staining. The interaction of KLF4/lncRNA-ZFAS1 promoter and lncRNA-ZFAS1/FTO was measured by ChIP, dual-luciferase reporter, or RIP assays. The m6A levels of Drp1 were measured by MeRIP-PCR. Mitochondrial staining and transmission electron microscopy (TEM) were used to evaluate the mitochondrial morphology in N2a cells and brain tissues. BMSC-derived exosomes increased the viability of neuronal cells treated with OGD/R while decreasing LDH release, oxidative stress, mitochondrial injury, and apoptosis. Furthermore, these effects were abolished by knockdown of exosomal KLF4. KLF4 increased lncRNA-ZFAS1 through binding to its promoter. LncRNA-ZFAS1 overexpression suppressed the m6A levels of Drp1 and reversed the promoting effect of exosomal KLF4 silencing on mitochondrial injury and the imbalance of mitochondrial dynamics by targeting FTO. Exosomal KLF4 alleviated the infarct area, neuronal injury, and apoptosis in MCAO mice through lncRNA-ZFAS1/FTO/Drp1 axis. BMSC-derived exosomal KLF4 promoted lncRNA-ZFAS1 expression to repress Drp1 m6A modification by targeting FTO, thus reducing mitochondrial dysfunction and alleviating neuronal injury in ischemic stroke.

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Acknowledgements

We would like to give our sincere gratitude to the reviewers for their constructive comments.

Funding

This work was supported by the National Natural Science Foundation of China, “Study on Long Noncoding RNA Regulating the Differentiation Direction of Neural Stem Cells” (project number: 81760234).

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  1. Department of Neurosurgery, Haikou Affiliated Hospital of Central South University Xiangya School of Medicine, No. 43, People’s Avenue, Haidian Island, Haikou, 570208, Hainan Province, People’s Republic of China

    Qing-Song Wang, Rong-Jun Xiao, Jun Peng, Zheng-Tao Yu, Jun-Qi Fu & Ying Xia

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Contributions

Qing-Song Wang: conceptualization, writing—original draft preparation, investigation, validation, visualization, and methodology. Rong-Jun Xiao: methodology and data curation. Jun Peng: software. Zheng-Tao Yu: data curation. Jun-Qi Fu: software. Ying Xia: conceptualization, writing—original draft preparation, supervision, and writing—reviewing and editing.

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Correspondence to Ying Xia.

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Supplementary Figure 1.

The function of exosomal KLF4 and lncRNA-ZFAS1 on the oxidative stress, mitochondrial injuries and apoptosis of primary cultured neurons. (A) The expression of KLF4 in primary cultured neurons co-treated with OGD/R and different concentration of exosomes was measured by Western blotting. (B) The viability of primary cultured neurons was analyzed by MTT assay after transfecting with Exo-shKLF4 or co-transfecting with Exo-shKLF4 and ZFAS1 overexpressing vector. (C-E) ELISA kits were performed to detect the levels of ROS, MDA and GSH-Px in primary cultured neurons transfected with Exo-shKLF4 or co-transfected with Exo-shKLF4 and ZFAS1 overexpressing vector. (F) The changes of MMP in OGD/R primary cultured neurons transfected with Exo-shKLF4 or co-transfected with Exo-shKLF4 and ZFAS1 overexpressing vector were assessed by JC-1 assay. (G and H) The apoptosis of OGD/R primary cultured neurons transfected with Exo-shKLF4 or co-transfected with Exo-shKLF4 and ZFAS1 overexpressing vector and the expression of apoptosis-related proteins were measured by flow cytometry and Western blotting. * p<0.05, ** p<0.01, *** p<0.001. (PNG 1422 kb)

High resolution image (TIF 69342 kb)

Supplementary Figure 2.

The effect of exosomal KLF4 and lncRNA-ZFAS1 axis on FTO expression and the balance of mitochondrial dynamics in primary cultured neurons. (A-D) Relative expression of Drp1, Fis1, OPA1 and Mfn1 in primary cultured neurons transfected with Exo-shKLF4 or co-transfected with Exo-shKLF4 and ZFAS1 overexpressing vector was measured by qRT-PCR. (E and F) The expression of FTO at mRNA and protein levels in primary cultured neurons transfected with Exo-shKLF4 or co-transfected with Exo-shKLF4 and ZFAS1 overexpressing vector was evaluated by qRT-PCR and Western blotting. * p<0.05, ** p<0.01, *** p<0.001. (PNG 405 kb)

High resolution image (TIF 3011 kb)

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Wang, QS., Xiao, RJ., Peng, J. et al. Bone Marrow Mesenchymal Stem Cell-Derived Exosomal KLF4 Alleviated Ischemic Stroke Through Inhibiting N6-Methyladenosine Modification Level of Drp1 by Targeting lncRNA-ZFAS1. Mol Neurobiol 60, 3945–3962 (2023). https://doi.org/10.1007/s12035-023-03301-2

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