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. 2021 Mar;41(3):530-545.
doi: 10.1177/0271678X20916860. Epub 2020 Apr 4.

Rbfox-1 contributes to CaMKIIα expression and intracerebral hemorrhage-induced secondary brain injury via blocking micro-RNA-124

Fang Shen  1   2 , Xiang Xu  1 , Zhengquan Yu  1 , Haiying Li  1 , Haitao Shen  1 , Xiang Li  1 , Meifen Shen  1   2 , Gang Chen  1
Affiliations

Rbfox-1 contributes to CaMKIIα expression and intracerebral hemorrhage-induced secondary brain injury via blocking micro-RNA-124

Fang Shen et al. J Cereb Blood Flow Metab. 2021 Mar.

Abstract

RNA-binding protein fox-1 homolog 1 (Rbfox-1), an RNA-binding protein in neurons, is thought to be associated with many neurological diseases. To date, the mechanism on which Rbfox-1 worsens secondary cell death in ICH remains poorly understood. In this study, we aimed to explore the role of Rbfox-1 in intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI) and to identify its underlying mechanisms. We found that the expression of Rbfox-1 in neurons was significantly increased after ICH, which was accompanied by increases in the binding of Rbfox-1 to Ca2+/calmodulin-dependent protein kinase II (CaMKIIα) mRNA and the protein level of CaMKIIα. In addition, when exposed to exogenous upregulation or downregulation of Rbfox-1, the protein level of CaMKIIα showed a concomitant trend in brain tissue, which further suggested that CaMKIIα is a downstream-target protein of Rbfox-1. The upregulation of both proteins caused intracellular-Ca2+ overload and neuronal degeneration, which exacerbated brain damage. Furthermore, we found that Rbfox-1 promoted the expression of CaMKIIα via blocking the binding of micro-RNA-124 to CaMKIIα mRNA. Thus, Rbfox-1 is expected to be a promising therapeutic target for SBI after ICH.

Keywords: CaMKIIα; Rbfox-1; intracerebral hemorrhage; micro-RNA-124; secondary brain injury.

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Conflict of interest statement

Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Intracerebral hemorrhage (ICH) model and experimental design. (a) Representative whole brains and coronal brain sections at the scheduled times after ICH. (b) Time course of the protein level and relationship between RNA-binding protein, fox-1 homolog 1 (Rbfox-1), and Ca2+/calmodulin-dependent protein kinase II (CaMKIIα) after ICH in vivo. (c) The effect of Rbfox-1 on ICH-induced injury. (d) The protein levels of Rbfox-1 and CaMKIIα after ICH in vitro and the potential mechanisms.
Figure 2.
Figure 2.
The protein levels of Rbfox-1 and CaMKIIα and the mRNA level of CaMKIIα in brain tissues after ICH. (a) Western blot analysis and quantification of the time course of the protein levels of Rbfox-1 in brain tissue around hematoma, n = 6. (b) Double-immunofluorescence analysis was performed with Rbfox-1 antibodies (green) and a neuronal marker (NeuN, red) in sections. Additionally, nuclei were fluorescently labeled with 4ʹ-6-diamidino-2-phenylindole (DAPI) (blue). Representative images of the Sham and ICH (12 h) groups are shown. Arrows pointed to Rbfox-1-positive neurons. Scale bar = 50 μm. (c) Co-immunoprecipitation (Co-IP) and RNA Immunoprecipitation (RIP) test analysis of the interaction between Rbfox-1 and CaMKIIα mRNA in brain tissues of rats at 12 h after ICH, n = 3. (d) Western blot analysis and quantification of the time course of the protein levels of CaMKIIα in brain tissue around hematoma, n = 6. In (a, d), mean values for the Sham group were normalized to 1.0. All data are mean ± standard deviations (SD). *P < 0.05, **P < 0.01 vs. Sham; #P < 0.05 vs. 6 h.
Figure 3.
Figure 3.
The effects of overexpression and knockdown of Rbfox-1 on the Rbfox-1, CaMKIIα mRNA, and CaMKIIα levels in brain tissues, and neurological function of rats after ICH. (a) The most effective siRNA was screened by 293 T cells. (b) Western blot analysis and quantification of the effects of plasmid and siRNA of Rbfox-1, n = 6. (c) The mRNA levels of CaMKIIα in brain tissue after overexpression and knockdown of Rbfox-1 were analyzed by polymerase chain reaction (PCR), n = 3. (d) Western blot analysis and quantification of the effects of overexpression and knockdown of Rbfox-1 on the protein levels of CaMKIIα in brain tissue, n = 6. (e) Neurobehavioral scores. (f) Adhesive-removal test. (g) Rotarod test. In (b–d), mean values for the Sham group were normalized to 1.0, data are mean ± SD. **P < 0.01 vs. Sham; #P < 0.05 vs. ICH + Vector; $$P < 0.01vs. ICH + Si-NC. In (e), the data are median and interquartile range; in (f, g), data are mean ± SD. ***P < 0.001 vs. Sham, #P < 0.05, ##P < 0.01 vs. ICH + Vector, $P < 0.05, $$P < 0.01 vs. ICH + Si-NC, n = 10.
Figure 4.
Figure 4.
The effects of overexpression and knockdown of Rbfox-1 on neuronal death and degeneration in brain tissues. (a) Double staining for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (green) neuronal marker (NeuN, red). Nuclei were fluorescently labeled DAPI (blue). Arrows point to TUNEL-positive cells, Scale bar = 50 μm. The percentage of TUNEL-positive cells is shown (b). (c) Pearson correlation coefficient between CaMKIIα levels with percentage of TUNEL-positive neurons in brain tissues within 12 h after ICH. R2=0.6214, P < 0.001, n = 36. (d) Positive fluoro-Jade B (FJB) staining and arrows point to FJB-positive cells. Scale bar = 200 μm. Counts of FJB-positive cells in brain cortex and perihematoma brain are shown (e, f). In (b, e, f), data are mean ± SD, ***P < 0.001 vs. Sham; ##P < 0.01 vs. ICH + Vector; $$P < 0.01 vs. ICH + Si-NC, n = 6.
Figure 5.
Figure 5.
The protein levels of Rbfox-1 and CaMKIIα in neurons after oxyhemoglobin (OxyHb) treatment and overexpression and knockdown of Rbfox-1. (a, b) Western blot analysis and quantification of the time course of the protein levels of Rbfox-1 and CaMKIIα in cultured neurons. (c, d) Western blot analysis and quantification of the effects of overexpression and knockdown of Rbfox-1 on the protein levels of Rbfox-1and CaMKIIα in cultured neurons. In (b, d), mean values for the control group were normalized to 1.0. Data are mean ± SD. (b) **P < 0.01 vs. Control; ##P < 0.01 vs. 6 h, n = 3. (d) **P < 0.01 vs. Control; #P < 0.05 vs. OxyHb + Vector; $$P < 0.01 vs. OxyHb + Si-NC, n = 3.
Figure 6.
Figure 6.
The effects of overexpression and knockdown of Rbfox-1 on OxyHb-induced neuronal apoptosis. (a, b) Western blot analysis and quantification of the effects of overexpression and knockdown of Rbfox-1 on the protein levels of cleaved caspase-12, bax, and bcl-2 in cultured neurons, n = 3. (c, d) Annexin V and PI double staining and flow-cytometry analysis showed neuronal apoptosis in various groups in vitro. PI−/Annexin V+ and PI+/Annexin V+ indicated apoptotic neurons, n = 3. (e) Ca2+ staining was used to analyze intracellular Ca2+ concentration in cultured neurons. Scale bar = 50 μm. All data are mean ± SD. In (b), mean values for the control group were normalized to 1.0. **P < 0.01 vs. Control; #P < 0.05, ##P < 0.01 vs. OxyHb + Vector; $P < 0.05, $$P < 0.01 vs. OxyHb + Si-NC. In (d), *P < 0.05 vs. Control; ##P < 0.01 vs. OxyHb + Vector; $P < 0.05 vs. OxyHb + Si-NC.
Figure 7.
Figure 7.
The roles of micro-RNA-124 (miR-124) with Rbfox-1 and CaMKIIα, and the roles of Rbfox-1 in SBI after ICH. (a) The co-localization of Rbfox-1 and CaMKIIα mRNA in cultured neurons after OxyHb treatment. Double staining for CaMKIIα mRNA (red) by Fish and Rbfox-1 (green) by immunofluorescence. And nuclei were fluorescently labeled with DAPI (blue). Scale bar = 50 μm. (b, c) The competition of Rbfox-1 and miR-124 on CaMKIIα expression. 293 T cells were co-transfected with miR-124 mimics or miR-124 inhibitors with or without over-Rbfox-1 or si-Rbfox-1 for 48 h. Western-blot analysis and quantification of the protein levels of Rbfox-1 and CaMKIIα in 293 T cells, n = 3. In (c), mean values for the control group were normalized to 1.0. Data are mean ± SD. *P < 0.05 vs. Control group; $P < 0.05 vs. miR-124 mimic group; #P < 0.05 vs. miR-124 inhibitor group. (d) Double staining for CaMKIIα mRNA (red) by FISH and Argonaute2 (AGO2) (green) in 293 T cells by immunofluorescence. Scale bar = 50 μm. (e) The mechanism of Rbfox-1 in SBI after ICH. After ICH, Rbfox-1 could compete with miR-124 for CaMKIIα mRNA to stabilize the CaMKIIα mRNA, and promote CaMKIIα mRNA expression, which in turn leaded to overload of Ca2+ and activated the apoptosis program.
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References

    1. Saand AR, Yu F, Chen J, et al.. Systemic inflammation in hemorrhagic strokes – a novel neurological sign and therapeutic target? J Cereb Blood Flow Metab 2019; 39: 959–988. - PMC - PubMed
    1. Shen H, Liu C, Zhang D, et al.. Role for RIP1 in mediating necroptosis in experimental intracerebral hemorrhage model both in vivo and in vitro. Cell Death Dis 2017; 8: e2641. - PMC - PubMed
    1. Lee L, Lo YT, See AAQ, et al.. Long-term recovery profile of patients with severe disability or in vegetative states following severe primary intracerebral hemorrhage. J Crit Care 2018; 48: 269–275. - PubMed
    1. Zhu MX, Lu C, Xia CM, et al.. Simvastatin pretreatment protects cerebrum from neuronal injury by decreasing the expressions of phosphor-CaMK II and AQP4 in ischemic stroke rats. J Mol Neurosci 2014; 54: 591–601. - PubMed
    1. Colbran RJ.Targeting of calcium/calmodulin-dependent protein kinase II. Biochem J 2004; 378: 1–16. - PMC - PubMed

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