RESUMO
Cerebral infarction (CI) has become a leading cause of death in China. Long non-coding RNAs (lncRNAs) are intensively involved in the progression of CI. Here, we aimed to investigate the effects of lncRNA LOC366613 (LOC366613) on cerebral I/R injury, as well as its possible mechanism. Transient middle cerebral artery occlusion (MCAO) was used to establish a mouse model of cerebral I/R, and the PC12 cell line was used to establish an in vitro oxygen-glucose deprivation (OGD) injury model. The MTT assay was used to determine cell viability, and qRT-PCR was used to determine RNA levels. Western blotting was conducted to detect protein expression levels. The TUNEL assay and flow cytometry were used to measure cell apoptosis, and 2,3,5-triphenyltetrazolium chloride (TTC) was used to determine cerebral infarct volume. Finally, RNA pull-down and luciferase activity assays were used to examine interactions between miR-532-5p and LOC366613, as well as between miR-532-5p and phosphatase and tensin homolog (PTEN). LOC366613 was overexpressed in patients with cerebral I/R injury. In PC12 cells, knockdown of LOC366613 reduced the apoptosis rate and lactic acid dehydrogenase (LDH) expression, while increasing cell viability. Moreover, miR-532-5p was shown to be a target of LOC366613, as predicted. Downregulation of miR-532-5p reversed the effects of LOC366613 knockdown on PC12 cell apoptosis, LDH release, and cell viability. Finally, PTEN was verified as a target of miR-532-5p. LOC366613 participates in cerebral I/R injury by regulating the miR-532-5p/PTEN axis, potentially providing a new CI treatment target.
Assuntos
Isquemia Encefálica/genética , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Sequência de Bases , Isquemia Encefálica/complicações , Isquemia Encefálica/patologia , Regulação para Baixo/genética , Glucose/deficiência , Humanos , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Oxigênio , Células PC12 , RNA Longo não Codificante/genética , Ratos , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Regulação para Cima/genéticaRESUMO
Background: Wnt/ß-catenin signaling has been reported to exert cytoprotective effects in a cellular model of Parkinson's disease (PD). Glutamate excitotoxicity has been suggested to contribute to the pathogenesis of PD, and excitatory amino acid transporters (EAATs) play a predominant role in clearing excessive glutamate. EAAT2 is mainly expressed in astrocytes, which are an important source of Wnt signaling in the brain. Methods: Wnt1-overexpressing U251 astrocytes were indirectly cocultured with dopaminergic SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA). Cell toxicity was determined by cell viability and flow cytometric detection. Glutamate level in the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). Western blot analysis was used to detect the expression of Wnt1, ß-catenin, and EAAT2. Immunofluorescence was used to display the expression and translocation of NF-κB p65. Results: 6-OHDA treatment significantly decreased cell viability in both U251 cells and SH-SY5Y cells, inhibited the expression of Wnt1, ß-catenin, and EAAT2 in U251 cells, and increased the glutamate level in the culture medium. Coculture with Wnt1-overexpressing U251 cells attenuated 6-OHDA-induced apoptosis in SH-SY5Y cells. Overexpression of Wnt1 decreased the glutamate level in the culture media, upregulated ß-catenin, EAAT2, and NF-κB levels, and promoted the translocation of NF-κB from the cytoplasm to the nucleus in U251 cells. Conclusion: Wnt1 promoted EAAT2 expression and mediated the cytoprotective effects of astrocytes on dopaminergic cells. NF-κB might be involved in the regulation of EAAT2 by Wnt1.