Ji Liang1, Qiang Wang1, Jun-Qi Li1, Tie Guo2, Dan Yu3. 1. Department of Neurology, Affiliated Haikou Hospital of Xiangya Medical College, Central South University, Haikou 570208, Hainan Province, PR China. 2. Department of ICU, The First Affiliated Hospital of Zhengzhou University, PR China. 3. Department of Neurology, Affiliated Haikou Hospital of Xiangya Medical College, Central South University, Haikou 570208, Hainan Province, PR China. Electronic address: yudanyuyue@163.com.
Abstract
OBJECTIVE: Inflammasome contributes to ischemic brain injury by inducing pyroptosis and inflammation. The aim of this study is to unravel the mechanism of long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3)-mediated regulation of absent in melanoma 2 (AIM2) inflammasome during cerebral ischemia/reperfusion (I/R). METHODS: In vivo middle cerebral artery occlusion (MCAO) rat model and in vitro oxygen-glucose deprivation/reperfusion (OGD/R)-treated neurocytes model were generated. TTC, H&E staining and TUNEL were performed to assess the cerebral ischemic injury. LDH and MTT assays were used to detect cell viability and cytotoxicity. qRT-PCR was used to detect the expression levels of MEG3, miR-485 and AIM2. Immunohistochemistry (IHC) and immunofluorescence were conducted to detect the AIM2 expression. ELISA and Western blotting were performed to determine the secretion and protein levels of inflammasome signaling proteins. Dual luciferase reporter assay and Ago2-RIP were used to validate the direct interaction among MEG3, miR-485 and AIM2. RESULTS: In both MCAO rats and OGD/R-treated neurocytes, MEG3 and AIM2 were significantly up-regulated, whereas miR-485 was down-regulated. MCAO induces pyroptosis and release of IL-1β and IL-18 in ischemia brain. MEG3 acted as a molecular sponge to suppress miR-485, and AIM2 was identified as a direct target of miR-485. Knockdown of MEG3 inhibited OGD/R-induced pyroptosis and inflammation, and lack of MEG3 inhibited caspase1 signaling and decreased the expression of AIM2, ASC, cleaved-caspase1 and GSDMD-N. While overexpression of MEG3 exerted opposite effects. CONCLUSION: MEG3/miR-485/AIM2 axis contributes to pyroptosis via activating caspase1 signaling during cerebral I/R, suggesting that this axis may be a potent therapeutic target in ischemic stroke.
OBJECTIVE: Inflammasome contributes to ischemic brain injury by inducing pyroptosis and inflammation. The aim of this study is to unravel the mechanism of long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3)-mediated regulation of absent in melanoma 2 (AIM2) inflammasome during cerebral ischemia/reperfusion (I/R). METHODS: In vivo middle cerebral artery occlusion (MCAO) rat model and in vitro oxygen-glucose deprivation/reperfusion (OGD/R)-treated neurocytes model were generated. TTC, H&E staining and TUNEL were performed to assess the cerebral ischemic injury. LDH and MTT assays were used to detect cell viability and cytotoxicity. qRT-PCR was used to detect the expression levels of MEG3, miR-485 and AIM2. Immunohistochemistry (IHC) and immunofluorescence were conducted to detect the AIM2 expression. ELISA and Western blotting were performed to determine the secretion and protein levels of inflammasome signaling proteins. Dual luciferase reporter assay and Ago2-RIP were used to validate the direct interaction among MEG3, miR-485 and AIM2. RESULTS: In both MCAOrats and OGD/R-treated neurocytes, MEG3 and AIM2 were significantly up-regulated, whereas miR-485 was down-regulated. MCAO induces pyroptosis and release of IL-1β and IL-18 in ischemia brain. MEG3 acted as a molecular sponge to suppress miR-485, and AIM2 was identified as a direct target of miR-485. Knockdown of MEG3 inhibited OGD/R-induced pyroptosis and inflammation, and lack of MEG3 inhibited caspase1 signaling and decreased the expression of AIM2, ASC, cleaved-caspase1 and GSDMD-N. While overexpression of MEG3 exerted opposite effects. CONCLUSION:MEG3/miR-485/AIM2 axis contributes to pyroptosis via activating caspase1 signaling during cerebral I/R, suggesting that this axis may be a potent therapeutic target in ischemic stroke.