Exosomal miR-370-3p increases the permeability of blood-brain barrier in ischemia/reperfusion stroke of brain by targeting MPK1

Abstract

Ischemia/reperfusion (I/R) damage induced by stroke poses a serious hazard to human life, while mechanism of blood-brain barrier (BBB) dysfunction is still unknown. To imitate stroke induced ischemia conditions in vivo, the rat model of cerebral I/R damage was created by middle cerebral artery occlusion (MCAO). In vitro, the rat microvascular endothelial cell line bEND.3 was subjected to oxygen-glucose deprivation/reperfusion (OGD/R). Evans blue was used to evaluate the permeability of the blood-brain barrier (BBB). To evaluate gene expression at the mRNA and protein levels, researchers used real-time PCR and western blotting. Infarct volume and BBB permeability were considerably higher in cerebral (I/R) animals than in the Sham group. Exosomal miR-370-3p expression was shown to be higher in the brains of I/R injured rats and OGD/R treatment bEND.3. The BBB permeability was considerably increased when miR-370-3p was downregulated in OGD/R pretreated bEND.3. miR-370-3p regulates MAPK1 expression by targeting it. In bEND.3, OGD/R therapy increased BBB permeability substantially. OGD/R was inhibited by miR-370-3p mimic transfection, while miR-370-3p mimic was abolished by co-transfection with MAPK1 overexpression lentivirus. In cerebral I/R damage, exosomal miR-370-3p targets MAPK1 and aggregates BBB permeability.

Keywords: blood-brain barrier; exosomes; ischemia/reperfusion; mir-370-3p; stroke.

Figure 1

Expression of miR-370-3p in the cerebral I/R rats. (A) Experimental design to measure BBB leakage in cerebral I/R rats (n=10). (B) Infarct volume was assessed using 2,3,5-triphenyltetrazolium chloride (TTC) staining. (C) BBB permeability was evaluated by Evan’s blue assay. (D) The top 5 most highly expressed mRNAs in I/R rats based on RNA sequencing data. (E) The relative expressions of miR-370-3p in the cerebral I/R rats were determined by qPCR. (F) miR-370-3p levels were assessed by immunofluorescent staining. (G) Correlation between miR-370-3p and infraction volume. (H) Correlation between miR-370-3p and BBB permeability. ** p<0.01, *** p<0.005, **** p<0.001.

Figure 2

Exosomes derived from cerebral I/R rats exhibit miR-370-3p upregulation. (A) The levels of CD63, TSG101, and α-Tubulin were tested by western blotting. (B) Cerebral MVs-derived exosomes were analyzed via electron microscopy (scale bar, 50 nm). (C) Relative miR-370-3p expression in exosomes derived from MVs were tested by qPCR. (D) After treatment with 2 mg/ml RNase alone or combined with 0.1% Triton X-100, miR-370-3p level was analysed using qPCR in the culture medium of MVs. (E) miR-370-3p expression was assessed by fluorescence in situ hybridization (FISH) in cerebral I/R rats and healthy rats. (F) miR-370-3p expression levels in bEND.3, were assessed via qPCR. (G) After treatment with 2 mg/ml RNase alone or combined with .1% Triton X-100, miR-370-3p level was analysed using qPCR in the culture medium of bEND.3. (H) Quantitative analysis indicated that relative fluorescence intensity in bEND.3. ** p<0.01, *** p<0.005, **** p<0.001.

Figure 3

GW4968 and Rab27a silencing down-regulate miR-370-3p in cerebral MVs. (A) Following co-culture with exosomes treated with GW4869, miR-370-3p was analyzed in bEND.3 cells. (B) After Rab27a silencing, miR-370-3p levels in bEND.3 cells were tested by qPCR. (C, D). miR-370-3p levels in bEND.3 cells were measured via qPCR following miR-370-3p mimic or inhibitor treatment. (E, F) Cerebral MVs from I/R rats were collected and then transfected with miR-370-3p mimic or inhibitor. miR-370-3p expression in exosomes from these cells then being assessed via qPCR. ns, not significant, ** p<0.01, *** p<0.005, **** p<0.001.

Figure 4

miR-370-3p aggravates I/R-induced BBB disruption. (A, B) The transendothelial electrical resistance of bEND.3 cells was measured using Millicell-ERS electrical resistance system. (C, D) The relative expressions of miR-370-3p in cerebral I/R rats with miR-370-3p mimics or inhibitors treatment. (E, F) Infraction volume and Evan blue leakage were used to evaluate BBB permeability with miR-370-3p mimics or inhibitor treatment. ns, not significant, ** p<0.01, *** p<0.005, **** p<0.001.

Figure 5

miR-370-3p directly targets MAPK1. (A) Candidate genes from three databases were shown in a Venn diagram. (B) Luciferase reporter constructs with WT or mutant (MUT) MAPK1 3'-UTR binding sites are shown schematically. (C) Following co-transfection of miR-370-3p mimics with WT or MUT reporter plasmids, luciferase activity in bEND.3 cells was assessed. (D) Interactions between MAPK1 and miR-370-3p were tested using RNA immunoprecipitation. (E, F) Relative MAPK1 expression in bEND.3 cells with miR-370-3p mimics or miR-370-3p inhibitor treatment. (G, H) Levels of MAPK1 in both cerebral I/R rats and OGD/R in bEND.3 cells.

Figure 6

miR-370-3p aggravated I/R-Induced BBB disruption by targeting MAPK1. (A) qPCR demonstrating that miR-370-3p regulates MAPK1 expression. (B) Transendothelial electrical resistance assessments was implemented to analyze BBB disruption. (C, D) Cellular proliferation following miR-370-3p mimic transfection and/or MAPK1 overexpression was assessed via infarct volume and BBB permeability in brain tissues of cerebral I/R rats. ** p<0.01, *** p<0.005, **** p<0.001.