Fan Zhao1, Yu Xing1, Pu Jiang1, Lai Hu1, Shixiong Deng2. 1. Department of Forensic Medicine, Chongqing Medical University, #1 Yixueyuan Road, Chongqing, 400016, China. 2. Department of Forensic Medicine, Chongqing Medical University, #1 Yixueyuan Road, Chongqing, 400016, China. Electronic address: dengshixiong1963@163.com.
Abstract
OBJECTIVE: The proliferation of neural stem cells (NSCs1), or lack thereof, can have profound effects on brain tissue remodeling for ischemic stroke (IS2). In this study, we aimed to reveal the influence of the lncRNA MEG3/miR-493-5p/MIF axis on NSC proliferation after IS. METHODS: We established an oxygen glucose-deprivation/reoxygenation (OGD/R3) in vitro model of IS in NSCs. We evaluated NSC isolation efficiency and proliferation by NESTIN, SOX2, and PCNA immunofluorescence staining. MEG3 and miR-493-5P levels were assessed by quantitative real-time polymerase chain reaction (qRT-PCR4). Changes in MIF protein expression levels were analyzed using Western blotting. We then evaluated the role of MEG3 and miR-493-5p by transfection of si-MEG3, a miR-493-5p mimic, or miR-493-5p inhibitor. NSC proliferation was quantified using Cell Counting Kit-8 analysis. RESULTS: NESTIN and SOX2 were co-expressed in endogenous NSCs. Following OGD/R, MEG3 and miR-493-5P were significantly upregulated in NSCs, while MIF levels decreased and proliferation was inhibited. Knockdown of MEG3 inhibited miR-493-5p and rescued expression of MIF and PCNA, restoring cellular proliferation levels. In NSCs transfected with a miR-493-5p mimic or inhibitor, MIF levels were down- or upregulated, respectively. Consistently, transfection of a miR-493-5p mimic reduced NSC proliferation, while transfection with a miR-493-5p inhibitor or si-MEG3 rescued the inhibitory effect of OGD/R on NSC proliferation. After co-transfection of si-MEG3 and a miR-493-5p mimic of OGD/R-induced NSCs, levels of PCNA, an indicator of cellular proliferation, were significantly reduced. Conclusion MEG3 inhibits NSC proliferation of after IS via positive regulation of miR-493-5p and potential subsequent downregulation of MIF.
OBJECTIVE: The proliferation of neural stem cells (NSCs1), or lack thereof, can have profound effects on brain tissue remodeling for ischemic stroke (IS2). In this study, we aimed to reveal the influence of the lncRNA MEG3/miR-493-5p/MIF axis on NSC proliferation after IS. METHODS: We established an oxygen glucose-deprivation/reoxygenation (OGD/R3) in vitro model of IS in NSCs. We evaluated NSC isolation efficiency and proliferation by NESTIN, SOX2, and PCNA immunofluorescence staining. MEG3 and miR-493-5P levels were assessed by quantitative real-time polymerase chain reaction (qRT-PCR4). Changes in MIF protein expression levels were analyzed using Western blotting. We then evaluated the role of MEG3 and miR-493-5p by transfection of si-MEG3, a miR-493-5p mimic, or miR-493-5p inhibitor. NSC proliferation was quantified using Cell Counting Kit-8 analysis. RESULTS: NESTIN and SOX2 were co-expressed in endogenous NSCs. Following OGD/R, MEG3 and miR-493-5P were significantly upregulated in NSCs, while MIF levels decreased and proliferation was inhibited. Knockdown of MEG3 inhibited miR-493-5p and rescued expression of MIF and PCNA, restoring cellular proliferation levels. In NSCs transfected with a miR-493-5p mimic or inhibitor, MIF levels were down- or upregulated, respectively. Consistently, transfection of a miR-493-5p mimic reduced NSC proliferation, while transfection with a miR-493-5p inhibitor or si-MEG3 rescued the inhibitory effect of OGD/R on NSC proliferation. After co-transfection of si-MEG3 and a miR-493-5p mimic of OGD/R-induced NSCs, levels of PCNA, an indicator of cellular proliferation, were significantly reduced. Conclusion MEG3 inhibits NSC proliferation of after IS via positive regulation of miR-493-5p and potential subsequent downregulation of MIF.
Authors: Carolina Estrada-Meza; Andrea Torres-Copado; Luisa Loreti González-Melgoza; Luis M Ruiz-Manriquez; Marcos De Donato; Ashutosh Sharma; Surajit Pathak; Antara Banerjee; Sujay Paul Journal: 3 Biotech Date: 2022-09-10 Impact factor: 2.893