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LncRNA C2dat2 facilitates autophagy and apoptosis via the miR-30d-5p/DDIT4/mTOR axis in cerebral ischemia-reperfusion injury.

ABSTRACT: Cerebral ischemia-reperfusion injury (CIRI) is an important pathophysiological process of ischemic stroke associated with various physiological and pathological processes, including autophagy and apoptosis. In this study, we examined the role and mechanism of long noncoding RNA CAMK2D-associated transcript 2 (C2dat2) in regulating CIRI in vivo and in vitro. C2dat2 up-regulation facilitated neuronal autophagy and apoptosis induced by CIRI. Mechanistically, C2dat2 acts as a competing endogenous RNA (ceRNA) to negatively regulate miR-30d-5p expression. More specifically, miR-30d-5p targeted the 3'-untranslated region of DNA damage-inducible transcript 4 (DDIT4) and silenced its target mRNA DDIT4. Additionally, C2dat2 binding with heat shock cognate 70/heat shock protein 90 blocked RNA-induced silencing complex assembly to abolish the miR-30d-5p targeting of DDIT4 and inhibited miR-30d-5p to silence its target mRNA DDIT4. Further analysis showed that C2dat2 knockdown conspicuously inhibited the up-regulation of DDIT4 and Beclin-1 levels and LC3B II/I ratio and the down-regulation of P62 and phosphorylated mammalian target of rapamycin (mTOR)/mTOR and phosphorylated-P70S6K/P70S6K ratio in Neuro-2a cells after oxygen-glucose deprivation/reoxygenation. This study first revealed that C2dat2/miR-30d-5p/DDIT4/mTOR forms a novel signaling pathway to facilitate autophagy and apoptosis induced by CIRI, contributing to the better understanding of the mechanisms of CIRI and enriching the ceRNA hypothesis in CIRI.

SUBMITTER: Xu Q

PROVIDER: S-EPMC8109078 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The increasing rates of morbidity and mortality caused by ischemic heart disease pose a serious threat to human health. Long non‑coding (lnc)RNA small nucleolar RNA host gene 1 (SNHG1) has a protective effect on the myocardium. In the present study, the role of lncRNA SNHG1 in myocardial ischemia reperfusion injury (MIRI) and the underlying mechanisms were investigated. After hypoxia/reoxygenation (H/R) induction, the expression levels of lncRNA SNHG1 were detected using reverse transcription‑quantitative PCR. After lncRNA SNHG1 overexpression via cell transfection, cell viability was detected using an MTT assay, apoptotic rates were detected using TUNEL staining, apoptosis‑related protein expression levels were detected using western blotting and respective kits were used to measure the oxidative stress levels. The Encyclopedia of RNA Interactomes database predicted the presence of binding sites between lncRNA SNHG1 and microRNA (miR)‑450b‑5p, and between miR‑450b‑5p and insulin‑like growth factor 1 (IGF1). These interactions were then verified using luciferase reporter assays. Subsequently, the regulatory mechanism underlying the lncRNA SNHG1/miR‑450b‑5p/IGF1 axis in MIRI was investigated by overexpressing miR‑450b‑5p and knocking down IGF1 expression in H/R‑induced cells. Finally, the expression of PI3K/Akt signaling pathway‑related proteins was detected using western blotting. lncRNA SNHG1 expression was significantly downregulated in H/R‑induced AC16 cells. lncRNA SNHG1 overexpression significantly inhibited apoptosis and decreased oxidative stress levels in H/R‑induced AC16 cells, which was mediated via regulation of the miR‑450b‑5p/IGF1 axis and activation of the PI3K/Akt signaling pathway. Therefore, the present study suggested that activation of the PI3K/Akt signaling pathway via the lncRNA SNHG1/miR‑450b‑5p/IGF1 axis inhibited the apoptosis and oxidative stress levels of H/R‑induced AC16 cells.

Project description:BackgroundIschemia stroke is the leading cause of disability, which is a consequence of vascular occlusion. The purpose of this study is to investigate the effect of cornin which is isolated from the fruit of Verbena officinalis L, against astrocytes autophagy induced by cerebral ischemia/reperfusion (CI/R) injury in vitro and in vivo and its potential mechanism.MethodsCornin at dose of 2.5, 5 and 10 mg/kg were intravenously injected to MCAO rats at 15 min after reperfusion. The infarction volume, blood-brain barrier (BBB), neurological severity score (mNSS), and autophagy related protein were used to evaluated the protective effects and potential mechanism of cornin in autophagy with or without phosphoinositide-3 kinase (PI3K)inhibitor LY294002 and mammalian target of rapamycin (mTOR) small interfering RNA (siRNA) at 24 h after CI/R injury. The potential protective effects and mechanism of cornin at concention of 10 ~ 1000 nM were also evaluated in oxygen glucose deprivation/reperfusion (OGD/R) in U87 cells.ResultsThe results suggest that cornin at dose of 5 or 10 mg/kg significantly reduce the cerebral infarction volume and blood-brain barrier (BBB) leakage, and improve neurological recovery in MCAO rats. Cleaved caspase-3 and Bax levels were significantly decreased, while B-cell lymphoma-2 (Bcl-2) and the apoptosis regulator ratio (Bcl-2/Bax) were markedly increased when treated with 2.5-10 mg/kg cornin. The obvious decreased expressions of glial fibrillary acidic protein (GFAP), myosin-like BCL2 interacting protein (Beclin-1) and microtubule-associated protein light chain 3 II (LC3-II) and increased of neuronal nuclei (NeuN), sequestosome-1 (p62), phosphorylated mTOR (p-mTOR), and phosphorylated Akt (p-Akt) were observed in MCAO rats treated with 10 mg/kg cornin, which was counteracted by LY294002. The expression of autophagy-related proteins with or without LY294002 and mTOR siRNA presented the similar results as in vitro in OGD/R in U87 cells.ConclusionsThese results indicate that cornin improved neurological recovery after cerebral ischemia injury by preventing astrocytes autophagy induced by CI/R via the PI3K/Akt/mTOR signaling pathway.

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LncRNA C2dat2 facilitates autophagy and apoptosis via the miR-30d-5p/DDIT4/mTOR axis in cerebral ischemia-reperfusion injury.

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