Yi Lu1, Yao Han1, Jinlin He1, Binbin Zhou1, Pu Fang1, Xiaobing Li2. 1. Department of Neurology, First Affiliated Hospital Nanchang University, Jiangxi Province, 330006, PR China. 2. Department of Neurology, First Affiliated Hospital Nanchang University, Jiangxi Province, 330006, PR China. Electronic address: xiaobingli1969@163.com.
Abstract
AIMS: Long non-coding RNAs (lncRNAs) FOXD3-AS1 was reported to be increased in cardiomyocyte ischemic injury. However, its role and underlying molecular mechanism in ischemic stroke remain unknown. This study was to investigate the role of FOXD3-AS1 in cerebral ischemia/reperfusion injury. METHODS: The expression of FOXD3-AS1 and miR-765 were measured with qRT-PCR. The shared putative miR-765 binding sites both in BCL2L13 and FOXD3-AS1 were identified with bioinformatics, luciferase reporter assay and RNA immunoprecipitation. Apoptosis and its related proteins were detected by TUNEL assay, Hoechst 33,258 staining, flow cytometry and western blot. Infarct volume and the neurological status were evaluated with TTC staining and neurologic deficit score, respectively. RESULTS: The up-regulation of FOXD3-AS1 and down-regulation of miR-765 were found in both mouse brains after cerebral ischemia/reperfusion (I/R) and neuroblastoma cells of neuro-2A (N2a) after oxygen-glucose deprivation/reoxygenation (OGD/R). Moreover, the overexpression of miR-765 reduced N2a cell apoptosis caused by OGD/R. MiR-765 could target BCL2L13 directly. In addition, we found that FOXD3-AS1 bound to miR-765 directly, acting as a ceRNA to modulate the expression of BCL2L13. Overexpression of FOXD3-AS1 antagonized the inhibitory impact of miR-765 on the expression of BCL2L13 and the apoptosis of N2a cells treated with OGD/R, while FOXD3-AS1 knockdown promoted the inhibitory impact of miR-765 on the expression of BCL2L13 and the apoptosis of N2a cells treated with OGD/R. Furthermore, we found that neurological deficits and brain injury induced by I/R in vivo were attenuated by FOXD3-AS1 knockdown. CONCLUSIONS: We verified a critical signaling pathway of FOXD3-AS1/miR-765/BCL2L13 in regulating cerebral ischemia/reperfusion injury.
AIMS: Long non-coding RNAs (lncRNAs) FOXD3-AS1 was reported to be increased in cardiomyocyte ischemic injury. However, its role and underlying molecular mechanism in ischemic stroke remain unknown. This study was to investigate the role of FOXD3-AS1 in cerebral ischemia/reperfusion injury. METHODS: The expression of FOXD3-AS1 and miR-765 were measured with qRT-PCR. The shared putative miR-765 binding sites both in BCL2L13 and FOXD3-AS1 were identified with bioinformatics, luciferase reporter assay and RNA immunoprecipitation. Apoptosis and its related proteins were detected by TUNEL assay, Hoechst 33,258 staining, flow cytometry and western blot. Infarct volume and the neurological status were evaluated with TTC staining and neurologic deficit score, respectively. RESULTS: The up-regulation of FOXD3-AS1 and down-regulation of miR-765 were found in both mouse brains after cerebral ischemia/reperfusion (I/R) and neuroblastoma cells of neuro-2A (N2a) after oxygen-glucose deprivation/reoxygenation (OGD/R). Moreover, the overexpression of miR-765 reduced N2a cell apoptosis caused by OGD/R. MiR-765 could target BCL2L13 directly. In addition, we found that FOXD3-AS1 bound to miR-765 directly, acting as a ceRNA to modulate the expression of BCL2L13. Overexpression of FOXD3-AS1 antagonized the inhibitory impact of miR-765 on the expression of BCL2L13 and the apoptosis of N2a cells treated with OGD/R, while FOXD3-AS1 knockdown promoted the inhibitory impact of miR-765 on the expression of BCL2L13 and the apoptosis of N2a cells treated with OGD/R. Furthermore, we found that neurological deficits and brain injury induced by I/R in vivo were attenuated by FOXD3-AS1 knockdown. CONCLUSIONS: We verified a critical signaling pathway of FOXD3-AS1/miR-765/BCL2L13 in regulating cerebral ischemia/reperfusion injury.