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

Abstract

MicroRNAs (miRNA) are short (~22nt) 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.

Fig. 1

Hspa5 is the target of miR-181. A, B. Dual luciferase activity assays performed in BOSC23 cells co-transfected with the plasmid containing Renilla luciferase followed by the Hspa1a or Hspa9 3′UTR (WT) and plasmids encoding either pri-miR-181ab or pri-miR-181cd or their seed mutants (SM) demonstrates that miR-181 does not recognize either of these 3′UTRs, as there is no reduction of luciferase activity compared to the SM control. C. The same assay performed with the Hspa5 3′UTR (Hspa5-WT) or its seed mutant (Hspa5-SM) shows that miR-181ab and cd both reduce luciferase activity. *P<0.05, statistically different from the SM group by T-test. Luciferase assays were performed 3 times in triplicate. D. Differing levels of expression of miR-181a, b, c, and d are detected in cortex of normal control brains. N=4 animals, assayed in triplicate, * P<0.01 different than levels of miR-181a by ANOVA and Newman-Keuls post hoc test.

Fig. 2

Expression of miR-181, GRP78 protein, and Grp78 mRNA after transient focal ischemia (MCAO). A. miR-181a expression in ischemic core and penumbra at different durations of reperfusion after 1 hr MCAO in mice shows increased levels in core but decreased levels in the penumbra (PNBR). B. miR-181b, c, and d show similar changes after ischemia, increasing in the core and decreasing in the penumbra. C. GRP78 protein decreases in the ischemic core and increases in the penumbra with increasing durations of reperfusion after MCAO. Quantitation by densitometry of westerns for 4 mice at each timepoint. D, E. Examples of western blots for GRP78 isolated from penumbra and ischemic core with increasing reperfusion time. F. Expression of Grp78 mRNA in ischemic core and penumbra increases with reperfusion time after MCAO. G, H. Examples of RT-PCR products from RNA harvested at increasing times after MCAO. Actin was used as the loading control for westerns and internal control for RT-PCR. N=4 mice/group in all experiments. *P<0.05 by ANOVA and Newman-Keuls post hoc test.

Fig. 3

Effect of pri-miR-181ab transfection on reactive oxygen species and ischemia-like astrocyte injury in vitro. A. pri-miR-181ab induced increased levels of miR-181a in astrocytes following transfection. B. pri-miR-181ab overexpression by plasmid transfection aggravates cell injury induced in primary astrocytes by 24 hr glucose deprivation (GD). Representative micrographs of cultures stained with PI and Hoechst dye are shown. C. Quantitation by cell counting is shown for 3 separate sets of cultures. D. Representative micrographs show increased ROS by increased HEt fluorescence in astrocytes after transfection with pri-miR-181a compared to its seed mutant (SM) control in unstressed cells under normal growth conditions. E. Bar graph shows quantitation of HEt fluorescence in transfected cultures under normal growth conditions. F. Representative micrograph shows similar mitochondrial membrane potential by TMRE fluorescence in cultures under normal growth conditions whether transfected with pri-miR-181ab or the seed mutant. G. Bar graph shows quantitation of TMRE fluorescence. All experiments were performed in triplicate using three sets of culture each time. *P<0.01 by T-test.

Fig. 4

Effect of miR-181a mimic and inhibitor on C6 glia with or without GRP78 overexpression. A. Dose-response of miR-181a levels to transfection with increasing amounts of miR-181a inhibitor in C6 cultures. N=3. The arrow indicates the concentration used for B, C, and D of this figure. B. Representative blot shows miR-181a inhibitor increases GRP78 protein expression both in parental C6 cells and in the C6 line that stably overexpresses His-Grp78 under the control of the CMV promoter and lacking the native 3′UTR. C. Bar graph showing quantitation of significant changes in GRP78 protein expression with miR-181a inhibitor. D. miR-181a inhibitor reduces injury induced by 8 hr glucose deprivation (GD) in C6 cultures and His-Grp78 overexpressing cultures. E. Dose-response of miR-181a levels after transfection with increasing amounts of mimic in C6 cultures. N=3. The arrows indicate the concentrations used for F, G, and H of this figure. F. Representative blot shows miR-181a mimic decreases GRP78 protein expression in both C6 and C6- His-Grp78 overexpressing cells. G. Bar graph shows quantitation of GRP78 protein expression after transfection with miR-181a mimic. Protein levels in Ctrl C6 cells and His-Grp78 overexpressing cells with 30 pmol mimic were not statistically different. N=3. H. miR-181a mimic aggravates injury induced by 8 hr GD in both C6 and His-GRP78 overexpressing C6 cells. Cell death in Ctrl C6 cells and His-Grp78 overexpressing cells with 30 pmol mimic were not statistically different. All experiments were performed 3 times in triplicate. *P<0.01 compared with same cell line control (Ctrl) and #P<0.01 compared with the same dose of miR-181 inhibitor or mimic in C6 group by ANOVA and Newman-Keuls post hoc test.

Fig. 5

Effect of miR-181 up- or down-regulation on miR-181a, GRP78, and infarction after focal ischemia. A. Mice injected ICV with pri-miR-181ab plasmid show elevated brain levels of miR-181a by RT-qPCR. B. Mice pretreated with miR-181a antagomir show reduced levels of miR-181a in the brain. C. GRP78 protein levels are decreased in the brains of mice pretreated with pri-miR-181ab plasmid. D. GRP78 protein levels increased in brains pretreated with miR-181a antagomir. Representative immunoblots are shown under the graphs. N=4 mice in each group for A to D. E. Representative cresyl violet-stained coronal sections demonstrate an increased infarct size in a representative miR-181a overexpressing brain compared with the brain of a miR-181 seed mutation (SM)-injected animal also subjected to MCAO. F. The bar graph shows quantitation of infarct size by cresyl violet staining for the group of mice. G. Representative cresyl violet stained brains of miR-181a antagomir transfected or mismatch (MM) miR-181a-antagomir-injected animals. H: The graph shows quantification of the infarct size. N=7 mice/group for E to H. *P<0.01 compared to SM or MM control by T-test.