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. 2021 Aug 13;13(15):19587-19597.
doi: 10.18632/aging.203369. Epub 2021 Aug 13.

LncRNA AC136007.2 alleviates cerebral ischemic-reperfusion injury by suppressing autophagy (VSports注册入口)

Na Liu  1 , Aini Peng  1 , Haiyan Sun  2 , Yuansu Zhuang  1 , Ming Yu  3 , Qun Wang  1 , Jinping Wang  1
Affiliations

"V体育官网" LncRNA AC136007.2 alleviates cerebral ischemic-reperfusion injury by suppressing autophagy

Na Liu et al. Aging (Albany NY). .

Abstract

Differential expression and diagnostic significance of the long noncoding RNA (lncRNA) AC136007. 2 has been reported in patients with acute ischemic stroke (AIS). However, its role on disease progression and outcome remains unclear. Here, we employed an oxygen-glucose deprivation/reperfusion (OGD/R) model in neuronal SH-SY5Y cells and performed middle cerebral artery occlusion (MCAO) surgery in rats to investigate the function of AC136007. 2 in ischemia-reperfusion (I/R) injury. AC136007. 2 expression was determined by RT-qPCR and cell viability was examined using CCK-8, Edu, LDH, and apoptosis assays. Pro-inflammatory cytokine expression was assessed using ELISA. OGD/R downregulated AC136007. 2 expression in SH-SY5Y cells, decreased viability by inducing apoptosis, and stimulated secretion of TNF-α, IL-6, and IL-1β. In turn, lentivirus-mediated AC136007 VSports手机版. 2 overexpression significantly reversed these phenomena. LC3 immunofluorescence and western blotting analyses of LC3-I/II and Beclin-1 expression and AMPK/mTOR phosphorylation status showed that AC136007. 2 suppressed autophagy in SH-SY5Y cells via inactivation of AMPK/mTOR signaling. Notably, incubation with the AMPK activator AICAR abolished the pro-survival effect of AC136007. 2 upon OGD/R treatment. Importantly, intraventricular injection of AC136007. 2 significantly reduced cerebral infarction and brain edema in MCAO rats, as shown by TTC staining and water content measurements. We conclude that AC136007. 2 alleviates cerebral I/R injury by suppressing AMPK/mTOR-dependent autophagy. .

Keywords: MCAO; OGD/R; autophagy; ischemic stroke; lncRNA V体育安卓版. .

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that there are no conflicts of interests regarding this manuscript.

Figures (VSports手机版)

Figure 1
Figure 1
Overexpression of AC136007.2 decreases OGD/R-induced cell death in SH-SY5Y cells. (A) Relative AC136007.2 expression level was evaluated by RT-qPCR in SH-SY5Y cells incubated in normoxic (control) conditions and after OGD/R treatment. (B) RT-qPCR analysis of AC136007.2 expression after transfection with lenti-AC136007.2 or its negative control. (C) Cell viability was determined by the CCK-8 assay. (D) Cell proliferation was determined by EdU assay. The rate of Edu-positive cells (histogram on the right side) was calculated from three random fields of view, with total cell numbers determined by DAPI staining. (E) LDH-based cytotoxicity analysis. **p<0.01 versus control group; ## p<0.01 versus OGD/R group; n = 3.
Figure 2
Figure 2
Overexpression of AC136007.2 decreases OGD/R-induced apoptosis and inflammatory cytokine release in SH-SY5Y cells. (A) Cell apoptosis was analyzed by flow cytometry after Annexin V/PI double staining. (B) Quantification of apoptosis rates from experiments like those shown in (A). (C–E) ELISA analysis of pro-inflammatory cytokine levels in cell culture supernatants. **p<0.01 versus control group; ## p<0.01 versus OGD/R group; n = 3.
Figure 3
Figure 3
Overexpression of AC136007.2 inhibits OGD/R-induced autophagy through inhibition of AMPK-mTOR signaling in SH-SY5Y cells. (A) Representative images of LC3 immunofluorescence and semi-quantitative analysis of autophagy levels (right). Cell nuclei were counterstained with DAPI. (B) Western blotting analysis of LC3 I/II, Beclin 1, p-AMPK, and p-mTOR expression and corresponding densitometric quantification data. Signals were normalized to β-actin. **p<0.01 versus control group; ## p<0.01 versus OGD/R group; ^^p<0.01 versus OGD/R + Lenti-AC136007.2 group; ^p<0.05 versus OGD/R + Lenti-AC136007.2 group; n = 3.
Figure 4
Figure 4
Autophagy inhibition mediates the cytoprotective effect of AC136007.2 against OGD/R-induced injury. (A) Cell viability was determined by the CCK-8 assay. (B) Cell apoptosis was analyzed by flow cytometry after Annexin V/PI double staining. **p<0.01 versus control group; ## p<0.01 versus OGD/R group; ^^p<0.01 versus OGD/R + Lenti-AC136007.2 group; n=3.
Figure 5
Figure 5
AC136007.2 reduces cerebral infarction, brain edema, and brain cell death after MCAO in rats. (A) Detection of brain infarction area by TTC staining. (B) TTC-based quantification of cerebral infarction (as percentage of the whole hemisphere). **p<0.01 versus I/R (MCAO) group; n=6. (C) Cerebral water content measurements. **p<0.01 versus I/R group; n=6. (D) TUNEL staining assessment of apoptosis in brain sections. **p<0.01 versus sham group; ## p<0.01 versus I/R group; n=6.
Figure 6
Figure 6
AC136007.2 inhibits IS-induced autophagy by inactivating AMPK-mTOR signaling. (A) Brain expression of LC3 I/II, Beclin 1, p-AMPK, and p-mTOR was evaluated by western blotting. (B–E) Quantification of western blotting results. Expression data were normalized to β-actin. **p<0.01 versus sham group; ## p<0.01 versus I/R group; n=6.
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