ΜicroRNA-122 protects against ischemic stroke by targeting Maf1

Abstract

The protection of brain tissue against damage and the reduction of infarct size is crucial for improving patient prognosis following ischemic stroke. Therefore, the present study aimed to investigate the regulatory effect of microRNA (miR)-122 and its target gene repressor of RNA polymerase III transcription MAF1 homolog (Maf1) on the infarct area in ischemic stroke. Reverse transcription-quantitative PCR (RT-qPCR) was performed to determine miR-122 expression levels in an ischemic stroke [middle cerebral artery occlusion (MCAO)] mouse model. Nissl staining was conducted to measure the infarct area of the MCAO mouse model. Moreover, RT-qPCR was performed to investigate the relationship between the expression of Maf1 and miR-122 in the MCAO mouse model. Dual-luciferase reporter assay in vitro and miR-122 mimic or inhibitor treatment in vivo were conducted to verify that miR-122 targeted and inhibited Maf1 expression. The results suggested that miR-122 was upregulated in the brain tissue of MCAO model mice. miR-122 overexpression effectively reduced the size of the infarct area in comparison with a control and miR-122 knockdown in brain tissue resulted in the opposite effect. Moreover, Maf1 was confirmed to be a direct target of miR-122. The results of a dual-luciferase reporter assay indicated that miR-122 bound to the 3'-untranslated region of Maf1. Maf1 expression decreased after stroke model induction in comparison with that in sham animals, and Maf1 expression was negatively associated with the expression of miR-122. In addition, miR-122 knockdown increased Maf1 expression levels, whereas miR-122 overexpression decreased Maf1 expression levels in comparison with a control. In conclusion, the results suggested that miR-122 improved the outcome of acute ischemic stroke by reducing the expression of Maf1.

Keywords: infarct area; ischemic stroke; microRNA-122; repressor of RNA polymerase III transcription MAF1 homolog.

Figure 1

MCAO model establishment. (A) The arrow indicates the middle cerebral artery which was not coagulated. (B) The arrow indicates the middle cerebral artery which was coagulated using electrocoagulation forceps. (C) Representative image of TTC-stained brain surface after ischemic stroke modeling. Pale areas indicate ischemic focal points. (D) Representative image of TTC-stained coronary surface after MCAO. Pale areas indicate ischemic focal points. MCAO, middle cerebral artery occlusion; TTC, 2,3,5-triphenyl tetrazolium chloride.

Figure 2

miR-122 overexpression in MCAO model rats effectively reduces the infarct area. (A) miR-122 expression in brain tissue. (B) Alterations to the infarct area after lateral ventricle injection of miR-122 NC, mimic or inhibitor. ^*P<0.05 vs. sham; ^#P<0.05 vs. control. miR, microRNA; MCAO, middle cerebral artery occlusion; NC, negative control.

Figure 3

Maf1 is a direct target of miR-122. (A) The sequence of miR-122/Maf1-WT-3'UTR/Maf1-MUT-3'UTR. Yellow represents the miR-122 and Maf1-WT-3'UTR function sites and the Maf1-MUT-3'UTR mutation sites. (B) Luciferase activity of 293A cells following transfection with miRNA and vector for 48 h. ^*P<0.05 vs. control. Ctrl, control; Maf1, repressor of RNA polymerase III transcription MAF1 homolog; miR, microRNA; WT, wild-type; UTR, untranslated region; MUT, mutant; NS, not significant.

Figure 3

Maf1 is a direct target of miR-122. (A) The sequence of miR-122/Maf1-WT-3'UTR/Maf1-MUT-3'UTR. Yellow represents the miR-122 and Maf1-WT-3'UTR function sites and the Maf1-MUT-3'UTR mutation sites. (B) Luciferase activity of 293A cells following transfection with miRNA and vector for 48 h. ^*P<0.05 vs. control. Ctrl, control; Maf1, repressor of RNA polymerase III transcription MAF1 homolog; miR, microRNA; WT, wild-type; UTR, untranslated region; MUT, mutant; NS, not significant.

Figure 4

miR-122 inhibits the expression of Maf1. (A) Maf1 expression levels in the sham operation and MCAO groups. (B) miR-122 expression in the brain tissue after lateral ventricle injection of miR-122 NC, mimic or inhibitor. (C) Maf1 expression levels in the brain tissue following injection of miR-122 NC, mimic or inhibitor into the lateral ventricle. ^*P<0.05 vs. sham; ^#P<0.05 vs. control. miR, microRNA; Maf1, repressor of RNA polymerase III transcription MAF1 homolog; NC, negative control.