LncRNA GAS5/miR-137 Is a Hypoxia-Responsive Axis Involved in Cardiac Arrest and Cardiopulmonary Cerebral Resuscitation

Abstract

Background: Cardiac arrest/cardiopulmonary resuscitation (CA/CPR) represents one of the devastating medical emergencies and is associated with high mortality and neuro-disability. Post-cardiac arrest syndrome (PCAS) is mechanistically ascribed to acute systemic ischemia/reperfusion(I/R) injury. The lncRNA/microRNA/mRNA networks have been found to play crucial roles in the pathogenesis of the hypoxia-responsive diseases. Nonetheless, the precise molecular mechanisms by which lncRNA/miRNA/mRNA axes are involved in the astrocyte-microglia crosstalk in CA/CPR have not been fully elucidated.

Methods: We collected and purified the exosomes from the blood of CA/CPR patients and supernatant of OGD/R-stimulated astrocytes. On the basis of microarray analysis, bioinformatic study, and luciferase activity determination, we speculated that lncRNA GAS5/miR-137 is implicated in the astrocyte-microglia crosstalk under the insult of systemic I/R injury. The regulation of lncRNA GAS5/miR-137 on INPP4B was examined by cellular transfection in OGD/R cell culture and by lateral ventricle injection with miR-137 agomir in CA/CPR mice model. Flow cytometry and immunofluorescence staining were performed to detect the microglial apoptosis, M1/M2 phenotype transformation, and neuroinflammation. Neurological scoring and behavior tests were conducted in CA/CPR group, with miR-137 agomir lateral-ventricle infusion and in their controls.

Results: In all the micRNAs, miR-137 was among the top 10 micRNAs that experienced greatest changes, in both the blood of CA/CPR patients and supernatant of OGD/R-stimulated astrocytes. Bioinformatic analysis revealed that miR-137 was sponged by lncRNA GAS5, targeting INPP4B, and the result was confirmed by Luciferase activity assay. qRT-PCR and Western blotting showed that lncRNA GAS5 and INPP4B were over-expressed whereas miR-137 was downregulated in the blood of CA/CPR patients, OGD/R-stimulated astrocytes, and brain tissue of CA/CPR mice. Silencing lncRNA GAS5 suppressed INPP4B expression, but over-expression of miR-137 negatively modulated its expression. Western blotting exhibited that PI3K and Akt phosphorylation was increased when lncRNA GAS5 was silenced or miR-137 was over-expressed. However, PI3K and Akt phosphorylation was notably suppressed in the absence of miR-137, almost reversing their phosphorylation in the silencing lncRNA GAS5 group. Then we found that GAS5 siRNA or miR-137 mimic significantly increased cell viability and alleviated apoptosis after OGD/R injury. Furthermore, over-expression of miR-137 attenuated microglial apoptosis and neuroinflammation in CA/CPR mice model, exhibiting significantly better behavioral tests after CA/CPR.

Conclusion: LncRNA GAS5/miR-137 may be involved in the astrocyte-microglia communication that inhibits PI3K/Akt signaling activation via regulation of INPP4B during CA/CPR.

Keywords: astrocyte; cardaic arrest; cardiopulmonary cerebral resuscitation; crosstalk; exosome; microglia; neuroinflammation.