MiR-139 protects against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced nerve injury through targeting c-Jun to inhibit NLRP3 inflammasome activation.

Cerebral ischemia/reperfusion (I/R) injury after ischemic stroke is usually accompanied with the activation of inflammasome which seriously impairs neurological function. MiR-139 has been reported to be associated with inflammatory regulation in multiple diseases. However, its effect and mechanism on inflammation regulation after cerebral I/R injury are still poorly understood. An in vitro model of cerebral I/R injury was constructed with oxygen-glucose deprivation/reoxygenation (OGD/R) treatment. TargetScan bioinformatics analysis and dual luciferase reporter assay were utilized to confirm the targeted relationship between miR-139 and c-Jun. Cell pyroptosis was verified by flow cytometry and Caspase-1 Detection Kit. qRT-PCR assay was performed to detect the expression levels of miR-139, c-Jun, NLRP3 and ASC. Western blotting was applied to measure the protein levels of c-Jun and pyroptosis-related markers NLRP3, ASC, caspase-1, GSDMDNterm. The ELISA assay was applied to measure the release of IL-1β, IL-18 and LDH. MiR-139 was significantly downregulated whereas c-Jun was obviously upregulated after OGD/R treatment. TargetScan analysis predicted that c-Jun was a potential target of miR-139, which was verified by the dual-luciferase reporter assay. Also, overexpression of miR-139 repressed c-Jun expression. Furthermore, miR-139 inhibited OGD/R-induced cell pyroptosis and the upregulation of NLRP3, caspase-1, ASC, GSDMDNterm, and the release of IL-1β, IL-18 and LDH, while miR-139 inhibition exerted the opposite effects. However, overexpression of c-Jun aggravated OGD/R-induced nerve injury and partly abolished the neuroprotective effect of miR-139. Upregulation of miR-139 exerted neuroprotection against OGD/R-induced nerve injury by negatively regulating c-Jun/NLRP3 inflammasome signaling. This study offered insights for providing potential therapeutic targets for treating cerebral I/R injury.

Wang QS ，Luo XY ，Fu H ，Luo Q ，Wang MQ ，Zou DY ... -  《-》

Long non-coding RNA MEG3 promotes cerebral ischemia-reperfusion injury through increasing pyroptosis by targeting miR-485/AIM2 axis.

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). 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. 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. 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.

Liang J ，Wang Q ，Li JQ ，Guo T ，Yu D ... -  《-》

LncRNA Gm44206 Promotes Microglial Pyroptosis Through NLRP3/Caspase-1/GSDMD Axis and Aggravate Cerebral Ischemia-Reperfusion Injury.

Yang L ，Gao Y ，Huang J ，Yang H ，Zhao P ，Li C ，Yang Z ... -  《-》

Lipopolysaccharide (LPS) Aggravates High Glucose- and Hypoxia/Reoxygenation-Induced Injury through Activating ROS-Dependent NLRP3 Inflammasome-Mediated Pyroptosis in H9C2 Cardiomyocytes.

Qiu Z ，He Y ，Ming H ，Lei S ，Leng Y ，Xia ZY ... -  《-》

LncRNA Tug1 Contributes Post-stroke NLRP3 Inflammasome-Dependent Pyroptosis via miR-145a-5p/Tlr4 Axis.

Pyroptosis, a type of programmed cell death illuminated by inflammasomes and active caspases, is implicated in post-stroke inflammation. Our previous study showed that lncRNA taurine upregulated gene 1 (Tug1) sponging miR-145a-5p modulated microglial activation after oxygen-glucose deprivation (OGD). However, the role and mechanism of Tug1 on post-stroke pyroptosis is not fully clear. Photo-thrombosis stroke mice and OGD-treated BV-2 microglia were established respectively. Tug1 knockdown or overexpression was achieved by intraventricular infusion of AAV-shTug1 in vivo, or transfection of siTug1 and pcDNA3.1-Tug1 in vitro. Neurological function and infarction volume were evaluated. Meanwhile, pyroptosis-associated proteins (IL-1β, IL-18, NLRP3, ASC, cleaved-caspase-1, and GSDMD-N), TLR4, and p-p65/p65 as well as Tug1 and miR-145a-5p were detected 24 h after photo-thrombosis or 4 h after OGD by qRT-PCR, western blot, and ELISA. The correlation between Tug1/miR-145a-5p/Tlr4 axis and pyroptosis was explored by dual-luciferase reporter assay and functional gain-and-loss experiments. Photo-thrombosis or OGD caused neural injury and upregulated pyroptosis-associated proteins, Tug1, TLR4, and p-p65 as well as downregulated miR-145a-5p, which was prevented by Tug1 knockdown in vivo and in vitro. Tlr4 gene, putatively binding with miR-145a-5p by bioinformatics analysis, was found to be a direct target of miR-145a-5p with negative interactions. Furthermore, miR-145a-5p inhibitor abolished the inhibitive effects of siTug1 on TLR4 and p-p65 as well as pyroptosis-associated proteins, whereas miR-145a-5p mimics abrogated the enhanced effects of pcDNA3.1-Tug1 on that, suggesting an involvement of Tug1/miR-145a-5p/Tlr4 axis on pyroptosis. Tug1 contributes NLRP3 inflammasome-dependent pyroptosis through miR-145a-5p/Tlr4 axis post-stroke, providing a promising therapeutic strategy against inflammatory injury.

Yao M ，Luo Y ，Li H ，Liao S ，Yu J ... -  《-》