The protective effects of lncRNA ZFAS1/miR-421-3p/MEF2C axis on cerebral ischemia-reperfusion injury

Abstract

LncRNA ZNFX1 antisense RNA 1 (ZFAS1) could improve neuronal damage and inhibit inflammation and apoptosis. We conducted an in-depth exploration on the protective mechanism of ZFAS1 in cerebral ischemia-reperfusion injury. Overexpressed or silenced plasmids of ZFAS1 were transfected into the cells to analyze the effects of oxygen-glucose deprivation/reperfusion (OGD/R) treatment on the viability, apoptosis and related gene expressions of Neuro-2a cell by performing MTT assay, flow cytometry, qRT-PCR, and Western blot. Bioinformatic analysis, qRT-PCR, dual-luciferase reporter assay and RNA immunoprecipitation were used to screen and verify the miRNA(s) which could competitively bind with ZFAS1 and downstream mRNA(s) targeted by the miRNA(s). The effects of ZFAS1 and the above target miRNA(s) or gene(s) on the apoptosis of OGD/R-injured cells, apoptosis-related proteins, inflammatory factors and p65/IκBα pathway were further verified via the rescue test. The results from the middle cerebral artery occlusion (MCAO) mouse model in vivo were consistent with those from the cellular experiments. The expression of lncRNA ZFAS1 in OGD/R-injured cells was inhibited, and the up-regulation of ZFAS1 protected Neuro-2a cells. MiR-421-3p was predicted to be the target miRNA of ZFAS1 and could offset the protective effect of ZFAS1 overexpression on OGD/R-injured cells following its up-regulation. MEF2C, which was the downstream target gene of miR-421-3p, reversed the OGD/R-induced enhanced cell damage caused by miR-421-3p mimic when MEF2C was overexpressed. In in vivo studies, ZFAS1 overexpression reduced brain tissue infarction, apoptosis and gene regulation caused by MCAO, while miR-421-3p mimic had the opposite effect. Collectively, the regulation of lncRNA ZFAS1/miR-421-3p/MEF2C axis showed protective effects on cerebral ischemia-reperfusion injury.

Keywords: cerebral ischemia-reperfusion injury; lncRNA ZNFX1 antisense RNA 1; miR-421-3p; myocyte enhancer factor 2C.

Figure 1.

Overexpressed ZFAS1 enhanced the viability of OGD/R-modeled cells and reduced apoptosis by regulating apoptosis-related proteins, while silenced ZFAS1 had the opposite effect. (A) The transfection efficiency of overexpressed/silenced ZFAS1 was determined by qRT-PCR. (B) The effects of overexpressed/silenced ZFAS1 and OGD/R treatment on ZFAS1 expression in Neuro-2a cells were detected by qRT-PCR. GAPDH was an internal reference. (C) The effects of overexpressed/silenced ZFAS1 and OGD/R treatment on the viability of Neuro-2a cells were determined by MTT assay. (D-E) The effects of overexpressed/silenced ZFAS1 and OGD/R treatment on the apoptosis of Neuro-2a cells were measured via flow cytometry. (F-I) The effects of overexpressed/silenced ZFAS1 and OGD/R treatment on apoptosis-related proteins in Neuro-2a cells were detected by western blot. GAPDH was an internal reference. All experiments were repeated thrice to average (n = 3). ^{^^^}p < 0.001 vs NC; ^&&&p < 0.001 vs shNC; ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 vs Control; *p < 0.05, **p < 0.01, ***p < 0.001 vs OGD/R+ NC; ^##p < 0.01, ^###p < 0.001 vs OGD/R+ shNC. ZFAS1, lncRNA ZNFX1 antisense RNA 1; shZFAS1, silent ZFAS1; OGD/R, oxygen-glucose deprivation/reperfusion; qRT-PCR, quantitative real-time polymerase chain reaction; NC, negative control; shNC, silent negative control.

Figure 2.

MiR-421-3p was confirmed to bind with ZFAS1. (A) The intersected miRNAs between ZFAS1 target miRNAs predicted by starBase database (http://starbase.sysu.edu.cn/) and differential miRNAs (GSE97532 data set) of MCAO selected by GEO (http://www.ncbi.nlm.nih.gov/geo) were obtained through a Venn diagram. (B) The up-regulated miRNAs in the intersected miRNAs were further verified by qRT-PCR to determine their regulatory relationship with ZFAS1. U6 was an internal reference. (C) The starBase showed the binding sequence of miR-421-3p and ZFAS1. (D) The dual-luciferase reporter assay verified the binding sequence of miR-421-3p and ZFAS1. (E-F) The association of miR-421-3p and ZFAS1 was verified by RNA immunoprecipitation experiment. All the experiments were repeated three times to average (n = 3). **p < 0.01, ***p < 0.001 vs NC; ^###p < 0.001 vs shNC; ⁺⁺p < 0.01 vs MC; ^&&&p < 0.001 Ago2+ MC; ^{^^^}p < 0.001 vs IgG+M. MCAO, middle cerebral artery occlusion; MC, mimic control.

Figure 3.

Up-regulation of miR-421-3p reversed the effect of overexpressed ZFAS1 on reducing the apoptosis of OGD/R-modeled cells. (A) The transfection efficiency of miR-421-3p mimic was tested by qRT-PCR. (B) The effects of miR-421-3p mimic and overexpressed ZFAS1 on the expression of miR-421-3p in OGD/R-modeled cells were detected by qRT-PCR. U6 was an internal reference. (C-D) The effects of miR-421-3p mimic and overexpressed ZFAS1 on the apoptosis of OGD/R-modeled cells were measured by flow cytometry. All the experiments were repeated three times to average (n = 3). ^{^^^}p < 0.001 vs MC; ⁺⁺⁺p < 0.001 vs Control; **p < 0.01, ***p < 0.001 vs OGD/R+ NC+MC; ^###p < 0.001 vs OGD/R+ ZFAS1+ MC; ^&&p < 0.01 vs OGD/R+ ZFAS1 + M.

Figure 4.

Up-regulation of miR-421-3p reversed the regulation of apoptosis-related proteins and inflammatory factors in OGD/R-modeled cells by overexpressed ZFAS1.

Figure 5.

MEF2C was a downstream gene of miR-421-3p, which bound to miR-421-3p. (A) The intersected mRNAs of the target genes of miR-421-3p were predicted by starBase database (http://starbase.sysu.edu.cn/) and miRDB database (http://mirdb.org/), and the differential mRNAs (GSE97533 dataset) of MCAO screened by GEO (http://www.ncbi.nlm.nih.gov/geo) were obtained through the Venn diagram. (B) The regulatory relationship between the intersected mRNAs and miR-421-3p mimic was further verified by qRT-PCR. GAPDH was an internal reference. (C) The starBase database showed the binding sequence of miR-421-3p and MEF2C. (D) The dual-luciferase reporter assay verified the binding sequence of miR-421-3p and MEF2C. All the experiments were repeated three times to average (n = 3). ^p < 0.05, ^^p < 0.01, ^^^p < 0.001 vs MC. MEF2C, Myocyte enhancer factor 2C

Figure 6.

MEF2C was up-regulated by overexpressed ZFAS1, but was down-regulated by miR-421-3p mimic in OGD/R cells. (A) The effects of overexpressed ZFAS1 and miR-421-3p mimic on the mRNA expression of MEF2C in OGD/R-modeled cells were analyzed by qRT-PCR. GAPDH was an internal reference. (B-C) The effects of overexpressed ZFAS1 and miR-421-3p mimic on the protein expression of MEF2C in OGD/R-modeled cells were analyzed by Western blot. GAPDH was an internal reference. All the experiments were repeated three times to average (n = 3). ++p < 0.01, +++p < 0.001 vs Control; **p < 0.01, ***p < 0.001 vs OGD/R+ NC+MC; #p < 0.05, ###p < 0.001 vs OGD/R+ ZFAS1+ MC; &&&p < 0.001 vs OGD/R+ ZFAS1 + M

Figure 7.

Overexpressed MEF2C reversed the pro-apoptotic effect of miR-421-3p mimic. (A) The transfection efficiency of MEF2C was tested by qRT-PCR. (B) The effects of overexpressed MEF2C and miR-421-3p mimic on the mRNA expression of MEF2C in OGD/R-modeled cells were analyzed by qRT-PCR. GAPDH was an internal reference. (C-D) The effects of overexpressed MEF2C and miR-421-3p mimic on the protein expression of MEF2C in OGD/R-modeled cells were analyzed by western blot. GAPDH was an internal reference. (E-F) The effects of overexpressed MEF2C and miR-421-3p mimic on the apoptosis of OGD/R-modeled cells were analyzed by flow cytometry. All the experiments were repeated three times to average (n = 3). ^{^^^}p < 0.001 vs MC; ⁺⁺⁺p < 0.001 vs Control; ***p < 0.001 vs OGD/R+ MC+NC; ^###p < 0.001 vs OGD /R + M+ NC; ^&&&p < 0.001 vs OGD/R+ MC+MEF2C.

Figure 8.

Overexpressed MEF2C reversed the gene regulation effect of miR-421-3p mimic. (A-C) The effects of overexpressed MEF2C and miR-421-3p mimic on the expressions of apoptosis-related proteins in OGD/R-modeled cells were detected by western blot. GAPDH was an internal reference. (D) The effects of overexpressed MEF2C and miR-421-3p mimic on the expressions of inflammatory factors in OGD/R-modeled cells were tested by qRT-PCR. GAPDH was an internal reference. (E-H) The effects of overexpressed MEF2C and miR-421-3p mimic on the p65/IκBα pathway in OGD/R-modeled cells were detected by western blot. GAPDH was an internal reference. All experiments were repeated three times to average (n = 3). ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 vs Control; *p < 0.05, **p < 0.01, ***p < 0.001 vs OGD/R+ MC+NC; ^##p < 0.01, ^###p < 0.001 vs OGD/R + M+ NC; ^&&p < 0.01, ^&&&p < 0.001 vs OGD/R+ MC+MEF2C.

Figure 9.

Overexpressed ZFAS1 reduced brain tissue infarction and apoptosis caused by MCAO, while miR-421-3p mimic had the opposite effect. (A) The effects of overexpressed ZFAS1 or miR-421-3p on the expression of ZFAS1 in the brain tissues of MCAO-modeled mice were tested by qRT-PCR. GAPDH was an internal reference. (B) The effects of overexpressed ZFAS1 or miR-421-3p on the expression of miR-421-3p in the brain tissues of MCAO-modeled mice were detected by qRT-PCR. GAPDH was an internal reference. (C-D) The effects of overexpressed ZFAS1 or miR-421-3p on the infarct area in the brain tissues of MCAO-modeled mice were observed and analyzed by TTC staining. (E-F) The effects of overexpressed ZFAS1 or miR-421-3p on cell apoptosis in the brain tissues of MCAO-modeled mice were observed and analyzed by TUNEL staining. n = 5. ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 vs Sham; *p < 0.05, **p < 0.01, ***p < 0.001 vs MCAO+ZFAS1+ Scramble; ^##p < 0.01, ^###p < 0.001 MCAO+ZFAS1+ Scramble; ^&p < 0.05, ^&&p < 0.01, ^&&&p < 0.001 vs MCAO+ZFAS1+ miR421-3p Agomir.

Figure 10.

Overexpressed ZFAS1 reversed the gene regulation effect caused by MCAO, while miR-421-3p mimic had the opposite effect. (A-B) The effects of overexpressed ZFAS1 or miR-421-3p on the expression of MEF2C in the brain tissues of MCAO-modeled mice were detected by western blot. GAPDH was an internal reference. (C-E) The effects of overexpressed ZFAS1 or miR-421-3p on apoptosis-related proteins in the brain tissues of MCAO-modeled mice were determined via western blot. GAPDH was an internal reference. (F) The effects of overexpressed ZFAS1 or miR-421-3p on inflammatory factors in brain tissues of MCAO-modeled mice were tested by qRT-PCR. GAPDH was an internal reference. (G-J) The effects of overexpressed ZFAS1 or miR-421-3p on the p65/IκBα pathway in the brain tissues of MCAO-modeled mice were detected by western blot. GAPDH was an internal reference. n = 5. ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 vs Sham; *p < 0.05, **p < 0.01, ***p < 0.001 vs MCAO+ZFAS1+ Scramble; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 MCAO+ZFAS1+ Scramble; ^&p < 0.05, ^&&p < 0.01, ^&&&p < 0.001 vs MCAO+ZFAS1+ miR-421-3p Agomir.