Research Paper Volume 13, Issue 8 pp 11315—11335
lncRNA C2dat2 facilitates autophagy and apoptosis via the miR-30d-5p/DDIT4/mTOR axis in cerebral ischemia-reperfusion injury
- 1 Henan Provincial Engineering Laboratory of Insects Bio-Reactor, Nanyang Normal University, Nanyang 473000, China
- 2 Henan Provincial Nanyang Central Hospital, Nanyang 473000, China
- 3 School of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang 473000, China
Received: July 7, 2020 Accepted: November 20, 2020 Published: April 4, 2021https://doi.org/10.18632/aging.202824
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Copyright: © 2021 Xu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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.
C2dat2: CAMK2D-associated transcript 2; CCK-8: Cell Counting Kit-8; ceRNAs: competing endogenous RNAs; CIRI: cerebral ischemia-reperfusion injury; CNC network: the coding-noncoding gene coexpression network; DAPI: 4′,6-diamidino-2-phenylindole; DDIT4: DNA damage-inducible transcript 4; FISH: fluorescence in situ hybridization; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GO: Gene Ontology; HLB: hypotonic lysis buffer; HSC70: heat shock cognate 70; HSP90: heat shock protein 90; I/R: ischemia-reperfusion; lncRNA: long noncoding RNA; MCAO: middle cerebral artery occlusion; miRNAs: microRNAs; MUT: mutant; MWS: modified Wuarin-Schibler; N2a: Neuro-2a; NC: negative control; ncRNA: noncoding RNA; OGD/R: oxygen-glucose deprivation/reoxygenation; mTOR: mammalian target of rapamycin; p-mTOR: phosphorylated mTOR; Ad-C2dat2: overexpressed C2dat2; Ad-NC: negative control vector; p-P70S6K: phosphorylated P70S6K; PBS: phosphate-buffered saline; RACE: rapid amplification of cDNA ends; RISC: RNA-induced silencing complex; RT-qPCR: reverse transcription-quantitative polymerase chain reaction; siRNA: small interfering RNA; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; UTR: untranslated region; WT: wild-type.