MicroRNA 320a functions as a novel endogenous modulator of aquaporins 1 and 4 as well as a potential therapeutic target in cerebral ischemia

Abstract

Aquaporins facilitate efficient diffusion of water across cellular membranes, and water homeostasis is critically important in conditions such as cerebral edema. Changes in aquaporin 1 and 4 expression in the brain are associated with cerebral edema, and the lack of water channel modulators is often highlighted. Here we present evidence of an endogenous modulator of aquaporin 1 and 4. We identify miR-320a as a potential modulator of aquaporin 1 and 4 and explore the possibility of using miR-320a to alter the expression of aquaporin 1 and 4 in normal and ischemic conditions. We show that precursor miR-320a can function as an inhibitor, whereas anti-miR-320a can act as an activator of aquaporin 1 and 4 expressions. We have also shown that anti-miR-320a could bring about a reduction of infarct volume in cerebral ischemia with a concomitant increase in aquaporins 1 and 4 mRNA and protein expression.

FIGURE 1.

Expression of AQP1 and AQP4 in astrocytoma cell line transfected with anti- or pre-miR-320a. A, relative miR-320a expression in astrocytoma cell line transfected with anti- or pre-miR-320a at a concentration of 30 nm. B, relative mRNA expression of AQP1, AQP4 in astrocytoma cell line transfected with anti- or pre-miR-320a at a concentration of 30 nm. Statistical analyses were done using t tests. *, p < 0.05; **, p < 0.01 compared with respective negative controls. The data shown are the means ± S.D., n = 3. C, AQP1 and AQP4 protein expression in astrocytoma cell line transfected with anti- or pre-miR-320a. Changes observed in mRNA expression were reflected in protein expression as well. β-Actin was used as a loading control. D, AQP immunoreactivites in astrocytoma cells transfected with anti- or pre-miR-320a. Human astrocytes were co-transfected with AQP1 or AQP4 plasmids and anti- or pre-miR-320a. The cells were fixed and immunolabeled with either anti-AQP1 or anti-AQP4 antibodies (green) and nuclear stain DAPI (blue). Anti-miR-320a-treated cells showed increased immunoreactivity for AQP1 and AQP4, whereas pre-miR-320a-treated cells showed a reduction in immunoreactivity.

FIGURE 2.

Relative luminescence in plasmid constructs containing miR-320a target sites. miR-320a target regions in the 3′-UTR of AQP1 and AQP4 were identified using TargetScan and microRNA.org. These regions were cloned into luciferase reporter plasmids. Mutated 3′-UTR constructs were generated using site-directed mutagenesis. The miRNA recognition sites at cDNA corresponding to 3′-UTR (underlined) were mutated as follows: AQP1 (5′-CTGATTCCTCTCATTTAATTTGGCT-3′) and AQP4 (5′-TTGCCCCATAAGAGCAGTCGTCCGG-3′). The plasmid constructs together with anti- or pre-miR-320a were co-transfected into HeLa cells. Luciferase luminescence readings were obtained 48 h post-transfection. Relative luminescence was obtained by normalizing the values against control plasmids, pMIR-REPORT^TM without any 3′-UTR insert. Statistical analyses were done using t tests. *, p < 0.05; **, p < 0.01 compared with control. The data shown are the means ± S.D., n = 3.

FIGURE 3.

Expression of AQP1 and AQP4 in astrocytoma cells subjected to 6 h of oxygen and glucose deprivation (OGD). A, changes in AQP1 and AQP4 mRNA and protein expression in cells subjected to oxygen and glucose deprivation. Total cellular RNA and protein were used to quantify AQP1 and AQP4 levels. Up-regulation of AQP1 and AQP4 mRNA and protein (see inset) were observed. B, relative AQP expression in cells transfected with anti- or pre-miR-320a and subjected to 6 h of oxygen and glucose deprivation. The cells were transfected with anti- or pre-miR-320a 48 h prior to oxygen and glucose deprivation. All of the values were expressed relative to negative controls. Statistical analyses were done using t tests. *, p < 0.05; **, p < 0.01 compared with control. The data shown are the means ± S.D., n = 3. Ctrl, control.

FIGURE 4.

Analyses of brain sections of MCA occluded rats injected with anti- or pre-miR-320a. A, expression level of miR-320a in ischemic rat injected with anti- or pre-miR-320a. Changes in miR-320a expression levels in the brain samples were quantitated using stem-loop real time quantitative PCR. B, histological analysis of brain sections. 2,3,5-Triphenyltetrazolium chloride-stained coronal brain sections (2 mm thick) of rats injected with 50 pmol of anti- or pre-miR-320a. Intracerebroventricular injections were given immediately after the removal of the suture (n = 10). Surviving cells stained red, whereas dead cells remained white. C, infarct volume of rat treated with anti- or pre-miR-320a. Infarct volumes are expressed as percentages of the control ± S.E. D, AQP1 and AQP4 mRNA levels in rats injected with anti- or pre-miR-320a. The data shown are the means ± S.D., n = 10. mRNA expression correlated with protein expression as well (see inset). E, relative AQP1, AQP4, and miR-320a expression in MK-801 administered ischemic (MCAo) rats. The rats with MCA occlusion were injected with MK-801 to reduce cerebral infarct. All of the values were expressed relative to ischemic rats. Statistical analyses were done using t tests. **, p < 0.01. The data shown are the means ± S.D., n = 10. Ctrl, control.

FIGURE 5.

Possible pathways/genes affected by miR-320a modulation. Compilation of possible genes affected by miR-320a modulation was adapted from the KEGG Pathway Database. Green ovals, targets of miR-320a; green rectangles, other genes in pathway; pink ovals, secondary messenger; blue right arrows, direct interaction; dashed blue right arrows, indirect interaction; broken red right arrows, AQP interaction.