Abstract
Progranulin (PGRN) is a secreted protein expressed ubiquitously throughout the body, including the brain, where it localizes in neurons and is activated microglia. Loss-of-function mutations in the GRN gene are an important cause of familial frontotemporal lobar degeneration (FTLD). PGRN has a neurotrophic and anti-inflammatory activity, and it is neuroprotective in several injury conditions, such as oxygen or glucose deprivation, oxidative injury, and hypoxic stress. Indeed, we have previously demonstrated that hypoxia induces the up-regulation of GRN transcripts. Several studies have shown microRNAs (miRNAs) involvement in hypoxia. Moreover, in FTLD patients with a genetic variant of GRN (rs5848), the reinforcement of miR-659-3p binding site has been suggested to be a risk factor. Here, we report that miR-659-3p interacts directly with GRN 3′UTR as shown by luciferase assay in HeLa cells and ELISA and Western Blot analysis in HeLa and Kelly cells. Moreover, we demonstrate the physical binding between GRN mRNA and miR-659-3p employing a miRNA capture-affinity technology in SK-N-BE and Kelly cells. In order to study miRNAs involvement in hypoxia-mediated up-regulation of GRN, we evaluated miR-659-3p levels in SK-N-BE cells after 24 h of hypoxic treatment, finding them inversely correlated to GRN transcripts. Furthermore, we analyzed an animal model of asphyxia, finding that GRN mRNA levels increased at post-natal day (pnd) 1 and pnd 4 in rat cortices subjected to asphyxia in comparison to control rats and miR-659-3p decreased at pnd 4 just when GRN reached the highest levels. Our results demonstrate the interaction between miR-659-3p and GRN transcript and the involvement of miR-659-3p in GRN up-regulation mediated by hypoxic/ischemic insults.
Highlights
Progranulin and HypoxiaProgranulin (PGRN) is a 65 kDa secreted protein expressed ubiquitously throughout the body, including the brain, where it localizes in neurons and is activated (Daniel et al, 2003; Petkau et al, 2010; Matsuwaki et al, 2011)
To investigate the binding of miR-659-3p to either the C allele or T allele GRN transcript and to calculate the hybridization minimum free energy in both cases we analyzed both sequences with RNAhybrid4 (Rehmsmeier et al, 2004)
The in silico analysis confirmed that miR-6593p can bind the GRN messenger RNA (mRNA) C allele with a DeltaG of −23.5 kcal/mol the binding is stronger to the T allele transcript (DeltaG = −25.6 kcal/mol)
Summary
Progranulin (PGRN) is a 65 kDa secreted protein expressed ubiquitously throughout the body, including the brain, where it localizes in neurons and is activated (Daniel et al, 2003; Petkau et al, 2010; Matsuwaki et al, 2011). We described that hypoxia up-regulates PGRN in neuroblastoma cell lines suggesting that it could exert a protective role in the brain against hypoxic stress, one of the main risk factors involved in FTLD pathogenesis (Piscopo et al, 2010). Our previous study showed that perinatal hypoxia triggers an early and transient oxidative stress in rat brain, followed by a biphasic regulation of several molecules involved in anti-oxidant defenses, neuroprotection and brain development. The early up-regulation of such molecules is likely to represent an adaptive response of the brain to counteract the consequences of the hypoxic insult (Piscopo et al, 2008)
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