Qingyi Ma1, Chiranjib Dasgupta2, Guofang Shen2, Yong Li2, Lubo Zhang3. 1. The Lawrence D. Longo Center for Perinatal Biology, Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA, 92350, USA. qma@llu.edu. 2. The Lawrence D. Longo Center for Perinatal Biology, Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA, 92350, USA. 3. The Lawrence D. Longo Center for Perinatal Biology, Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA, 92350, USA. lzhang@llu.edu.
Abstract
BACKGROUND: Neonatal 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. METHODS: We 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. RESULTS: HI 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. CONCLUSIONS: The miR-210-TET2 axis regulates pro-inflammatory cytokine expression in microglia, contributing to neonatal HI brain injury.
BACKGROUND: Neonatal 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. METHODS: We 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. RESULTS: HI 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. CONCLUSIONS: The miR-210-TET2 axis regulates pro-inflammatory cytokine expression in microglia, contributing to neonatal HI brain injury.
Authors: Janet E Orrock; Karuna Panchapakesan; Gilbert Vezina; Taeun Chang; Kari Harris; Yunfei Wang; Susan Knoblach; An N Massaro Journal: Pediatr Res Date: 2015-12-30 Impact factor: 3.756
Authors: Selma O Algra; Kathelijne M Groeneveld; Alvin W L Schadenberg; Felix Haas; Fabiola C M Evens; Jenny Meerding; Leo Koenderman; Nicolaas J G Jansen; Berent J Prakken Journal: J Neuroinflammation Date: 2013-02-07 Impact factor: 8.322