Abstract
BackgroundNeonatal hypoxic-ischemic (HI) brain injury is a leading cause of acute mortality and chronic disability in newborns. Our previous studies demonstrated that HI insult significantly increased microRNA-210 (miR-210) in the brain of rat pups and inhibition of brain endogenous miR-210 by its inhibitor (LNA) provided neuroprotective effect in HI-induced brain injury. However, the molecular mechanisms underpinning this neuroprotection remain unclear.MethodsWe made a neonatal HI brain injury model in mouse pups of postnatal day 7 to uncover the mechanism of miR-210 in targeting the ten eleven translocation (TET) methylcytosine dioxygenase 2 that is a transcriptional suppressor of pro-inflammatory cytokine genes in the neonatal brain. TET2 silencing RNA was used to evaluate the role of TET2 in the neonatal HI-induced pro-inflammatory response and brain injury. MiR-210 mimic and inhibitor (LNA) were delivered into the brain of mouse pups to study the regulation of miR-210 on the expression of TET2. Luciferase reporter gene assay was performed to validate the direct binding of miR-210 to the 3′ untranslated region of the TET2 transcript. Furthermore, BV2 mouse microglia cell line was employed to confirm the role of miR-210-TET2 axis in regulating pro-inflammatory response in microglia. Post-assays included chromatin immunoprecipitation (ChIP) assay, co-immunoprecipitation, RT-PCR, brain infarct assay, and neurobehavioral test. Student’s t test or one-way ANOVA was used for statistical analysis.ResultsHI insult significantly upregulated miR-210, downregulated TET2 protein abundance, and increased NF-κB subunit p65 acetylation level and its DNA binding capacity to the interleukin 1 beta (IL-1β) promoter in the brain of mouse pups. Inhibition of miR-210 rescued TET2 protein level from HI insult and miR-210 mimic decreased TET2 protein level in the brain of mouse pups, suggesting that TET2 is a functional target of miR-210. The co-immunoprecipitation was performed to reveal the role of TET2 in HI-induced inflammatory response in the neonatal brain. The result showed that TET2 interacted with NF-κB subunit p65 and histone deacetylase 3 (HDAC3), a co-repressor of gene transcription. Furthermore, TET2 knockdown increased transcriptional activity of acetyl-p65 on IL-1β gene in the neonatal brain and enhanced HI-induced upregulation of acetyl-p65 level and pro-inflammatory cytokine expression. Of importance, TET2 knockdown exacerbated brain infarct size and neurological deficits and counteracted the neuroprotective effect of miR-210 inhibition. Finally, the in vitro results demonstrated that the miR-210-TET2 axis regulated pro-inflammatory response in BV2 mouse microglia cell line.ConclusionsThe miR-210-TET2 axis regulates pro-inflammatory cytokine expression in microglia, contributing to neonatal HI brain injury.
Highlights
Neonatal hypoxic-ischemic (HI) brain injury is a leading cause of acute mortality and chronic disability in newborns
HI upregulated miR-210, suppressed Ten eleven translocation methylcytosine dioxygenase 2 (TET2) expression, and increased p65 acetylation level and binding activity at IL1β promoter in the brain of mouse pups Neonatal HI insult was introduced in produced in 7day-old (P7) mouse pups with the ligation of the right common carotid artery followed by hypoxic (8% O2) treatment
MiR-210 repressed TET2 expression in the neonatal brain To determine the role of miR-210 on TET2 expression after neonatal HI brain injury, either miR-210
Summary
Neonatal hypoxic-ischemic (HI) brain injury is a leading cause of acute mortality and chronic disability in newborns. HIE is a leading cause of acute mortality and chronic disability in newborns [1,2,3,4,5] and is associated with shortterm medical complications and long-term mental and other neurological disorders with delayed clinical onset [6, 7]. Mounting preclinical evidence supports the significant role of inflammation in the neonatal brain with HI insult [17, 19], and inhibition of the early inflammatory phase in the setting of HI brain injury confers neuroprotection [20, 21]. Elevated levels of pro-inflammatory cytokines and chemokines have been found in the cerebrospinal fluid and/or blood of infants with HIE, which are associated with adverse neurological outcome and correlate strongly with the likelihood of cerebral palsy [22,23,24]
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