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. 2022 Jul;11(7):1199-1208.
doi: 10.21037/tp-22-259.

The protective effect of MiR-27a on the neonatal hypoxic-ischemic encephalopathy by targeting FOXO1 in rats

Qun Cai  1 , Xiaoqun Zhang  1 , Liyuan Shen  1 , Honghua Song  1 , Ting Wang  2
Affiliations

The protective effect of MiR-27a on the neonatal hypoxic-ischemic encephalopathy by targeting FOXO1 in rats

Qun Cai et al. Transl Pediatr. 2022 Jul.

Abstract

Background: Neonatal hypoxic-ischemic encephalopathy (HIE), a kind of hypoxic-ischemic brain damage caused by perinatal asphyxia, is the most crucial cause of neonatal death and long-term neurological dysfunction in children. We aimed to investigate the protective effects of micro (mi)R-27a on HIE in neonatal rats.

Methods: A rat model of neonatal HIE was constructed by modification of the Rice-Vannucci model. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to test the expressions of miR-27a, FOXO1 messenger RNA (mRNA), interleukin-1β (IL-1β) mRNA, and tumor necrosis factor-α (TNF-α) mRNA, and western blot was applied to test the expression of FOXO1. In order to overexpress miR-27a, an intracerebroventricular injection (i.c.v) of miR-27a mimic was administered. We adopted 2,3,5-triphenytetrazolium chloride (TTC) staining and brain water content measurement to test the effects of miR-27a on the infarcted volume and edema in brain after HIE. Flow cytometry (FCM) analysis was applied to test the effects of miR-27a on the infiltrated peripheral immune cells in the rat brains after HIE.

Results: We successfully established a rat model of neonatal HIE. It was revealed that the expressions of miR-27a decreased gradually after HIE, however, the expressions of FOXO1 mRNA increased. After injection of the miR-27a mimic, the expression of miR-27a in the rat HIE model brains was significantly upregulated, however, the expression of FOXO1 was robustly downregulated. Both TTC staining and brain water content showed that the infarcted volume and brain edema was markedly increased after HIE. Interestingly, the overexpression of miR-27a reduced the infarcted volume and edema induced by HIE. Additionally, RT-qPCR and FCM analysis showed that HIE lead to increases of IL-1β, TNF-α, and infiltrated immune cells. Overexpression of miR-27a could reduce the expressions of IL-1β mRNA and TNF-α mRNA, and the cell numbers of infiltrated peripheral macrophages and neutrophils in the brain.

Conclusions: MiR-27a plays protective roles by reducing infarct volume and brain edema, and inhibiting inflammatory factors and infiltrated peripheral immune cells by targeting FOXO1 in neonatal HIE rats.

Keywords: FOXO1; infarcted volume; infiltrated peripheral immune cells; inflammation; miR-27a.

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-22-259/coif). The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
The expressions of miR-27a and FOXO1 mRNA after HIE in neonatal rats were tested by RT-qPCR. (A) The expressions of miR-27a after HIE were tested by RT-qPCR. (B) The expressions of FOXO1 mRNA after HIE were tested by RT-qPCR. *, P<0.05 and **, P<0.01 versus sham group. mRNA, messenger RNA; HIE, hypoxic-ischemic encephalopathy; RT-qPCR, real-time quantitative polymerase chain reaction.
Figure 2
Figure 2
The expressions of miR-27a and FOXO1 after injection of miR-27a mimic post HIE in neonatal rats were tested by RT-qPCR and western blot. (A) The expressions of miR-27a were tested by RT-qPCR. (B) The expressions of FOXO1 mRNA were tested by RT-qPCR. (C) The expressions of FOXO1 were tested by western blot. *, P<0.05, and **, P<0.01 versus sham group. ##, P<0.01. mRNA, messenger RNA; HIE, hypoxic-ischemic encephalopathy; RT-qPCR, real-time quantitative polymerase chain reaction.
Figure 3
Figure 3
The effects of miR-27a on infarcted volume and edema in brain after HIE in neonatal rats. (A) The infarcted volume was measured by TTC staining. (B) The brain water content was measured by dry-wet method. **, P<0.01 versus sham group. #, P<0.05. HIE, hypoxic-ischemic encephalopathy; TTC, 2,3,5-triphenytetrazolium chloride.
Figure 4
Figure 4
The effects of miR-27a on the inflammatory factors IL-1β and TNF-α after HIE in neonatal rats. (A) The expressions of IL-1β mRNA were measured by RT-qPCR. (B) The expressions of TNF-α mRNA were measured by RT-qPCR. **, P<0.01 versus sham group. #, P<0.05 and ##, P<0.01. IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; HIE, hypoxic-ischemic encephalopathy; RT-qPCR, real-time quantitative polymerase chain reaction; mRNA, messenger RNA.
Figure 5
Figure 5
The effects of miR-27a on the infiltrated peripheral lymphocytes in brain after HIE in neonatal rats. (A) The gating strategy of FCM analysis for peripheral lymphocytes subpopulation (T, B, and NK cells). (B) T cells (CD45+CD3+CD19−). (C) B cells (CD45+CD3−CD19+). (D) NK cells (CD45+CD3−NK1.1+). HIE, hypoxic-ischemic encephalopathy; FCM, flow cytometry; NK, natural killer.
Figure 6
Figure 6
The effects of miR-27a on infiltrated peripheral monocytes/macrophages and neutrophils in brain after HIE in neonatal rats. (A) The gating strategy of FCM analysis for peripheral monocytes/macrophages and neutrophils. (B) Monocytes (CD45hi+CD11b+). (C) Macrophages (CD45hi+CD11b+F4/80+). (D) Neutrophils (CD45hi+CD11b+LY6G+). *, P<0.05, and **, P<0.01 versus sham group. #, P<0.05. HIE, hypoxic-ischemic encephalopathy; FCM, flow cytometry.
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