lncRNA PINK1-AS Aggravates Cerebral Ischemia/Reperfusion Oxidative Stress Injury through Regulating ATF2 by Sponging miR-203

Abstract

Ischemic stroke is a common disease that led to high mortality and high disability. NADPH oxidase 2- (NOX2-) mediated oxidative stress and long noncoding RNA have important roles in cerebral ischemia/reperfusion (CI/R) injury, whereas whether there is interplay between them remains to be clarified. This study was performed to observe the role of lncRNA PINK1-antisense RNA (PINK1-AS) in NOX2 expression regulation. An in vivo rat model (MCAO) and an in vitro cell model (H/R: hypoxia/reoxygenation) were utilized for CI/R oxidative stress injury investigation. The expression levels of lncRNA PINK1-AS, activating transcription factor 2 (ATF2), NOX2, and caspase-3 and the production level of ROS and cell apoptosis were significantly increased in CI/R injury model rats or in H/R-induced SH-SY5Y cells, but miR-203 was significantly downregulated. There was positive correlation between PINK1-AS expression level and ROS production level. PINK1-AS and ATF2 were found to be putative targets of miR-203. Knockdown of lncRNA PINK1-AS or ATF2 or the overexpression of miR-203 significantly reduced oxidative stress injury via inhibition of NOX2. Overexpression of lncRNA PINK1 significantly led to oxidative stress injury in SH-SY5Y cells through downregulating miR-203 and upregulating ATF2 and NOX2. lncRNA PINK1-AS and ATF2 were the targets of miR-203, and the lncRNA PINK1-AS/miR-203/ATF2/NOX2 axis plays pivotal roles in CI/R injury. Therefore, lncRNA PINK1-AS is a possible target for CR/I injury therapy by sponging miR-203.

Figure 1

The increased lncRNA PINK1-AS associates with CI/R oxidative stress injury in rats. (a) mRNA level of lncRNA PINK-AS1; (b) ROS level; (c) correlation analysis; (d) protein expression of caspase3; (e) caspase3 activity; (f) representative images of TUNEL/Hoechst double staining; (g) the number of TUNEL-positive cell. The correlation between groups was analyzed by Pearson's correlation coefficient. ^∗∗P < 0.01 vs. sham; ^∗∗∗P < 0.001 vs. sham.

Figure 2

PINK1-AS knockdown reduced oxidative stress in H/R-treated SH-Y5Y cells. (a) mRNA level of lncRNA PINK-AS1; (b) presentative images of ROS; (c) ROS level; (d) correlation analysis; (e) protein expression of caspase3; (f) caspase3 activity; (g) the detection of cell apoptosis by Hoechst staining. (h) percentage of cell apoptosis. The correlation between groups was analyzed by Pearson's correlation coefficient. ^∗∗P < 0.01 vs. control; ^∗∗∗P < 0.001 vs. control; ^##P < 0.01 vs. H/R; ^###P < 0.001 vs. H/R.

Figure 3

lncRNA PINK1-AS and ATF2 are targets of miR-203. (a) sketch map of bioinformatic prediction; (b, c) relative luciferase activity. ^∗∗P < 0.01 vs. +lnc-PINK1-WT and +NC miRNA.

Figure 4

miR-203 inhibits oxidative stress injury by targeting ATF2. (a) miR-203 expression (rat); (b) ATF2 mRNA expression (rat); (c) ATF2 protein expression (rat); (d) miR-203 expression (SH-SY5Y cell); (e) ATF2 mRNA (SH-SY5Y cell); (f) ATF2 protein expression (SH-SY5Y cell); (g) presentative images of ROS; (h) ROS level; (i) the expression level of caspase3 protein; (j) caspase3 activity; (k) the detection of cell apoptosis by Hoechst staining. (l) percentage of cell apoptosis. The correlation between groups was analyzed by Pearson's correlation coefficient. ^∗P < 0.01 vs. sham or control. ^∗∗P < 0.01 vs. control or sham; ^#P < 0.05 vs. H/R; ^##P < 0.01 vs. H/R; ^###P < 0.001 vs. H/R.

Figure 5

Inhibition of ATF2 ameliorates oxidative stress injury relating to NOX2 expression regulation. (a, c) mRNA level of NOX2; (b, d) protein expression of NOX2; (e) NOX enzyme activity; (f) representative images of ROS; (g) ROS level; (h) protein expression of caspase3; (i) caspase3 activity; (j) the detection of cell apoptosis by Hoechst staining; (k) percentage of cell apoptosis. ^∗P < 0.05 vs. control or sham; ^∗P < 0.05 vs. control or sham; ^∗∗P < 0.01 vs. control or sham; ^#P < 0.05 vs. H/R; ^##P < 0.01 vs. H/R.

Figure 6

Overexpression of lncRNA PINK1-AS results in oxidative stress injury by regulating the miR-203/ATF2/NOX2 axis in SH-SY5Y cells. (a) lncRNA PINK1-AS expression level; (b) miR-203 expression level; (c) ATF2 mRNA expression; (d) ATF2 protein expression; (e) NOX2 mRNA expression; (f) NOX2 protein expression; (g) NOX enzyme activity; (h) presentative images of ROS level; (i) ROS level; (j) the detection of cell apoptosis by Hoechst staining; (k) percentage of cell apoptosis. ^∗P < 0.05 vs. control or sham; ^∗∗P < 0.01 vs. control; ^∗∗∗P < 0.001 vs. control.

Figure 7

Sketch map of CI/R injury induced by lncRNA PINK1-AS. The expression of lncRNA PINK1-AS increased in brain tissues that have undergone CI/R injury. Highly expressed lncRNA PINK1-AS contributed to a low expression of miR-203 by acting as a sponge of miR-203. Low expression of miR-203 led to ATF2 expression upregulation. As a transcript factor of NOX2, highly expressed ATF2 contributed to high expression of NCF2 and NOX2, which led to ROS generation. Excessive ROS resulted in neuron cell apoptosis.