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

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.

LncRNA CEBPA-AS1 knockdown prevents neuronal apoptosis against oxygen glucose deprivation/reoxygenation by regulating the miR-455/GPER1 axis.

Ischemic stroke (IS) is a common nervous system disease, which is a major cause of disability and death in the world. In present study, we demonstrated a regulatory mechanism of CCAAT/enhancer binding protein-alpha antisense 1 (CEBPA-AS1) in oxygen glucose deprivation/reoxygenation (OGD/R)-induced SH-SY5Y cells, with a focus on neuronal apoptosis. CEBPA-AS1, miR-455, and GPER1 expressions were evaluated by using qRT-PCR and Western blotting. The binding relationship among CEBPA-AS1, miR-455, and GPER1 was determined by a dual luciferase reporter assay. Neuronal viability and apoptosis were examined using MTT and flow cytometry assays, followed by determination of apoptosis-related factors (caspase 3, caspase 8, caspase 9, Bax, and Bcl-2). CEBPA-AS1 and GPER1 levels were upregulated, and miR-455 level was downregulated in the cell model of OGD/R induced. CEBPA-AS1 knockdown increased SH-SY5Y viability and reduced OGD/R-induced apoptosis. CEBPA-AS1 could act as a sponge of miR-455, and CEBPA-AS1 knockdown was found to elevate miR-455 expression. miR-455 overexpression also promoted SH-SY5Y cell viability and rescued them from OGD/R-induced apoptosis by binding to GPER1. GPER1 overexpression or miR-455 inhibition reversed the anti-apoptotic effect of CEBPA-AS1 knockdown. These findings suggest a regulatory network of CEBPA-AS1/miR-455/GPER1 that mediates neuronal cell apoptosis in the OGD model, providing a better understanding of pathogenic mechanisms after IS.