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. 2019 Jun;28(6):723-738.
doi: 10.1177/0963689718817219. Epub 2018 Dec 4.

Detrimental Role of miRNA-144-3p in Intracerebral Hemorrhage Induced Secondary Brain Injury is Mediated by Formyl Peptide Receptor 2 Downregulation Both In Vivo and In Vitro

Weijian Fan  1   2 , Xiang Li  1 , Dongping Zhang  1 , Haiying Li  1 , Haitao Shen  1 , Yizhi Liu  1 , Gang Chen  1
Affiliations

Detrimental Role of miRNA-144-3p in Intracerebral Hemorrhage Induced Secondary Brain Injury is Mediated by Formyl Peptide Receptor 2 Downregulation Both In Vivo and In Vitro

Weijian Fan et al. Cell Transplant. 2019 Jun.

Abstract

Although microRNA-144-3p (miRNA-144-3p) has been shown to suppress tumor proliferation and invasion, its function in intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI) remains unclear. Thus, this study was designed to investigate the role of miRNA-144-3p in ICH. To accomplish this, we used adult male Sprague-Dawley rats to establish an in vivo ICH model by injecting autologous blood, while cultured primary rat cortical neurons were exposed to oxyhemoglobin (OxyHb) to mimic ICH in vitro. To examine the role of miRNA-144-3p in ICH-induced SBI, we used an miRNA-144-3p mimic and inhibitor both in vivo and in vitro. Following ICH induction, we found miRNA-144-3p expression to increase. Additionally, we predicted the formyl peptide receptor 2 (FPR2) to be a potential miRNA-144-3p target, which we validated experimentally, with FPR2 expression downregulated when miRNA-144-3p was upregulated. Furthermore, elevated miRNA-144-3p levels aggravated brain edema and neurobehavioral disorders and induced neuronal apoptosis via the downregulation of FPR2 both in vivo and in vitro. We suspected that these beneficial effects provided by FPR2 were associated with the PI3K/AKT pathway. We validated this finding by overexpressing FPR2 while inhibiting PI3K/AKT in vitro and in vivo. In conclusion, miRNA-144-3p aggravated ICH-induced SBI by targeting and downregulating FPR2, thereby contributing to neurological dysfunction and neural apoptosis via PI3K/AKT pathway activation. These findings suggest that inhibiting miRNA-144-3p may offer an effective approach to attenuating brain damage incurred after ICH and a potential therapy to improve ICH-induced SBI.

Keywords: apoptosis; formyl peptide receptor 2; intracerebral hemorrhage; microRNA-144-3p.

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Conflict of interest statement

Declaration of Conflicting Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Fig. 1.
Fig. 1.
Establishments of an ICH model and experimental flow map. (A) Representative images of brain cortical tissues with a hematoma after infusion with autologous blood or with saline for the sham group. (B) Luciferase report between formyl peptide receptor 2 (FPR2) and miRNA-144-3p in 293 T cells. Time-courses examining miRNA-144-3p and FPR2 expression in brain perihematoma regions. (C and D) The role of miRNA-144-3p in regulating FPR2 after ICH-induced secondary brain injury (SBI) and its potential mechanisms.
Fig. 2.
Fig. 2.
MiRNA-144-3p and FPR2 expression after ICH. Adult male Sprague-Dawley (SD) rats were induced with ICH and brain samples were collected at various time points. (A) Real-time PCR analysis of miRNA-144-3p expression in perihematoma regions and the associated capacity curve. (B) Luciferase reporter analysis examining miRNA-144-3p and an FPR2 with a mutant or wild type 3′-UTR. (C) Western blot analysis and quantification of FPR2 levels in brain tissue collected in proximity to the hematoma at various time points. (D) Immunofluorescent staining of brain sections from the sham and ICH groups, with anti-FPR2 antibody (green), anti-NeuN antibody (red), and DAPI (blue) staining. Representative sham and ICH images at 24 h. All mean values for the sham group were normalized to 1.0 and all data are shown as a mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. sham group; #P < 0.05, ##P < 0.01, and @ P < 0.05 as indicated in the figure; n = 6.
Fig. 3.
Fig. 3.
MiRNA-144-3p downregulated FPR2 expression and induced neurological deficits and brain edema. Rats were randomly divided into 8 groups (n = 24 per group): sham group, ICH group, ICH + miRNA-144-3p mimic group (negative control), ICH + miRNA-144-3p mimic group, ICH + miRNA-144-3p inhibitor group (negative control), ICH + miRNA-144-3p inhibitor group, ICH + control siRNA group, and ICH + FPR2 siRNA group. All vectors, siRNA or miRNAs, were transfected 48 h before ICH induction and then brain samples were collected. (A) Real-time quantification of miRNA-144-3p expression after utilizing an miRNA-144-3p mimic or inhibitor. (B) LXA4 quantification in CSF within these groups as determined by ELISA. (C) Western blot analysis of brain FPR2 levels following miRNA-144-3p mimic or inhibitor transfection, after ICH induction, when compared with the control group. (D and E) The impact of miRNA-144-3p on neurological dysfunction at 1 w post-ICH induction and the brain water content, which was estimated based on five sections per sample. Sham mean values were normalized to 1.0 and all data are displayed as a mean ± SEM (n = 6). *P < 0.05 and **P < 0.01 vs. sham group; #P < 0.05, ##P < 0.01, @P < 0.05, @@P < 0.01, $P < 0.05, and $$P < 0.01 vs. their corresponding negative control group, as indicated in the figure. (D) The neurological deficiencies were evaluated based on the criteria of Garcia assessments, n = 12. The data are shown as a median with an interquartile range, n = 12. *P < 0.05 and **P < 0.01 vs. the sham group; #P < 0.05, ##P < 0.01, @P < 0.05, @@P < 0.01, $P < 0.05, $$P < 0.01 vs. their corresponding negative control group.
Fig. 4.
Fig. 4.
MiRNA-144-3p induced neuronal death and degradation after ICH. (A) Fluoro-Jade B (FJB) staining of paraffin sections, with arrows indicating FJB-positive cells. (B) The quantification of FJB-positive cells per mm2. (C) TUNEL staining (green), with neurons strained with NeuN (red). Arrows indicate apoptotic neurons that are NeuN/TUNEL-positive cells. (D) Quantification of TUNEL-positive neuronal cells. In (B) and (D) data are shown as a mean ± SEM, n = 6. *P < 0.05 and **P < 0.01 vs. sham group; ##P < 0.01, @P < 0.05, $$P < 0.01 vs. their corresponding negative control group.
Fig. 5.
Fig. 5.
The effects of miRNA-144-3p and FPR2 on the PI3K/AKT signal pathway in vivo. (A and B) Western blot analysis of PI3 K, p-PI3 K, AKT, and p-AKT in brain tissue collected in proximity to the hematoma after being transfected with miRNA-144-3p and FPR2 siRNA. Data are shown as mean ± SEM, n = 6. **P < 0.01 vs. sham group, ##P < 0.01, @P < 0.05, and $P < 0.05.
Fig. 6.
Fig. 6.
The effect of miRNA-144-3p on OxyHb-induced ICH in neurons. Primary neurons were cultured and stimulated with 10 μM OxyHb. (A) Western blot analysis examining FPR2 expression levels in neurons treated by OxyHb at the indicated time points. Data are shown as mean ± SEM, n = 3. *P < 0.05 and **P < 0.01 vs. control group; #P < 0.05 and @P < 0.05 vs. 12 h. (B) Neurons were transfected with siRNA or plasmid and then stimulated with 10 μM OxyHb for 12 h. The control group was normalized to 1.0 and β-tubulin was used as a loading control. The data are displayed as a mean ± SEM (n = 3). **P < 0.01 vs. control group; ##P < 0.01, control group vs. miRNA-144-3p mimic group; and @P < 0.05, control group vs. miRNA-144-3p inhibitor group. (C) The apoptotic neurons were analyzed by flow cytometry, and the apoptotic index was quantified. The data are shown as a mean ± SEM (n = 3). **P < 0.01 vs. control group; ##P < 0.01, control group vs. miRNA-144-3p mimic group; and @@P < 0.01, control group vs. miRNA-144-3p inhibitor group.
Fig. 7.
Fig. 7.
The effects of miRNA-144-3p and FPR2 on the PI3K/AKT signaling pathway in vitro. Neurons were transfected with siRNA or plasmid, and then stimulated with 10 μM OxyHb for 12 h. The cells were then collected and lysed for Western blot analysis. (A) Quantification of PI3K and p-PI3K levels, with p-PI3K levels in the control group normalized to 1.0, while the others were normalized to PI3K. Data are shown as mean ± SEM (n = 3). **P < 0.01 vs. control group; ##P < 0.01, control group vs. miRNA-144-3p mimic group; and @P < 0.05, control group vs. miRNA-144-3p inhibitor group. (B) Quantification of AKT and p-AKT, with p-AKT levels in the control group normalized to 1.0, while the others were normalized to AKT. Data are shown as mean ± SEM (n = 3). **P < 0.01 vs. control group; ##P < 0.01, control group vs. miRNA-144-3p mimic group; and @@P < 0.01, control group vs. miRNA-144-3p inhibitor group. (C) Quantification of cleaved caspase-3, with protein level in the control group normalized to 1.0 and β-actin used as a loading control. Data are shown as mean ± SEM (n = 3). **P < 0.01 vs. control group; ##P < 0.01, control group vs. miRNA-144-3p mimic group; and @@P < 0.01, control group vs. miRNA-144-3p inhibitor group. (D) Neurons overexpressing FPR2 after OxyHb exposure and PI3K/AKT pathway inhibitor treatment. Cleaved caspase-3 levels were quantified and the data are shown as mean ± SEM (n = 3). **P < 0.01 vs. control group; ##P < 0.01, OxyHb group vs. Mk-2206 group.
Fig. 8.
Fig. 8.
Illustration of the mechanisms of miRNA-144-3p in ICH-induced SBI. Endogenous miRNA-144-3p is activated after stimulation from blood cells and the hematoma. The miRNA-144-3p then binds the FPR2 3′-UTR and subsequently downregulates its expression at the transcriptional phase. FPR2 exhibits a neural protective function by activating the PI3K/AKT signal pathway, which may subsequently suppress the caspase-3 cascade and inhibit apoptosis.
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