miR-181 regulates GRP78 and influences outcome from cerebral ischemia in vitro and in vivo

Abstract

MicroRNAs (miRNA) are short (~22 nt) single stranded RNAs that downregulate gene expression. Although recent studies indicate extensive miRNA changes in response to ischemic brain injury, there is currently little information on the roles of specific miRNAs in this setting. Heat shock proteins (HSP) of the HSP70 family have been extensively studied for their multiple roles in cellular protection, but there is little information on their regulation by miRNAs. We used bioinformatics to identify miR-181 as a possible regulator of several HSP70 family members. We validated GRP78/BIP as a target by dual luciferase assay. In response to stroke in the mouse we find that miR-181 increases in the core, where cells die, but decreases in the penumbra, where cells survive. Increased levels of miR-181a are associated with decreased GRP78 protein levels, but increased levels of mRNA, implicating translational arrest. We manipulated levels of miR-181a using plasmid overexpression of pri-miR-181ab or mimic to increase, and antagomir or inhibitor to reduce levels. Increased miR-181a exacerbated injury both in vitro and in the mouse stroke model. Conversely, reduced levels were associated with reduced injury and increased GRP78 protein levels. Studies in C6 cells show that if GRP78 levels are maintained miR-181a no longer exerts a toxic effect. These data demonstrate that miR-181 levels change in response to stroke and inversely correlate with levels of GRP78. Importantly, reducing or blocking miR-181a protects the brain from stroke.

Neuroprotection of microRNA in neurological disorders (Review)

“…In the murine stroke model, the expression of miR-181 increases where the cells die, the core, but decreases where the cells survive, the penumbra (38). The multiple roles of glucose-regulated protein (GRP)-78, a member of the heat-shock protein 70 family, in cellular protection is validated as a target of miR-181 by the dual luciferase assay.…”

microRNAs: Innovative Targets for Cerebral Ischemia and Stroke

“…Using this method, transient ischemia is achieved by inserting a suture into the carotid artery and advancing it to block the middle cerebral artery. After temporarily blocking blood flow to the middle cerebral artery territory for a predetermined duration of minutes to hours, depending on the specific study, the suture is removed to allow reperfusion [19,48,49]. In some cases permanent occlusion is used.…”

“…Primary brain cell cultures or acute or cultured slices generally from the hippocampus, are subjected to medium lacking glucose, and in the case of OGD, also placed in a chamber with very low oxygen levels for a fixed period of time [5,[8][9][10]19,50,51] followed by restoration of oxygen and glucose to the medium to imitate reperfusion. The advantage of cell culture is that individual cell types can be studied, while in the case of hippocampal slice a brain slice containing some intact circuitry and relatively intact anatomical connections is studied.…”

“…There are so far a handful of reports on miRNA in focal ischemia [15][16][17] and a single report in forebrain ischemia [18]. We recently reported that a highly brain-enriched miRNA, miR-181, targets both of these protective mechanisms -the chaperone and anti-apoptotic BCL2 family members BCL2 [14,19]. Studying miRNAs as part of the brain's response to ischemia, and defining targets and mechanisms underlying their effects, will both add critical knowledge to our understanding of miRNA function and identify promising new targets for the development of novel treatments for ischemic brain injury and neurodegenerative diseases.…”

Identification of Loop Nucleotide Polymorphisms Affecting MicroRNA Processing and Function

“…MiRNAs are reported to be involved in a wide range of biological activities, such as immunity (Chen et al, 2004;Li et al, 2007a), cancer (O'Donnell et al, 2005;Zhang et al, 2013), apoptosis (Xu et al, 2003), stem cell maintenance (Arnold et al, 2011;Gangaraju and Lin, 2009) and neurological diseases (Cheng et al, 2009;Ouyang et al, 2012a). MiRNAs are initially transcribed as part of a long primary transcript or primary-miRNA (pri-miRNA) in the nucleus and are further processed by a RNase III Drosha/DGCR8 complex to generate 60-70-nt-long hairpin precursor miRNAs (pre-miRNA) (Lee et al, 2003).…”