A Novel lncRNA ENST00000530525 Affects ANO1 Contributing to Blood-Brain Barrier Injury in Cultured hCMEC/D3 Cells Under OGD/R Conditions.

Abstract

Ischemic stroke (IS) is a major neurological disease with high fatality and residual disability burdens. Increasing amount of long noncoding RNAs (lncRNAs) have been revealed to play an important role in ischemic stroke. However, the roles and significances of most lncRNAs in ischemic stroke are still unknown.This study was performed to identify differentially expressed lncRNAs using a lncRNA microarray in whole blood samples of patients suffered from acute cerebral ischemia. Bioinformatics analyses including GO, KEGG pathway enrichment analysis, and proximity to putative stroke risk location analysis were performed. A novel lncRNA ENST00000530525 significantly decreased after ischemic stroke. Furthermore, we evaluated lncRNA ENST00000530525 expression in cultured hCMEC/D3 cells under oxygen-glucose deprivation/reoxygenation(OGD/R) conditions using fluorescent in situ hybridization (FISH) and quantitative real-time polymerase chain reaction (RT-qPCR) analysis. To investigate the function of lncRNA ENST00000530525, the plasmid of overexpression(OE) and negative control(NC) were transfected into hCMEC/D3, then cell viability was detected by cell counting kit-8 (CCK-8) Assay after OGD/R,lncRNA ENST00000530525 and ANO1 expression were investigated using RT-qPCR and Immunofluorescence. For blood-brain barrier(BBB) permeability,FITC-dextran transendothelial permeability assay and Tight junction(Tj) protein was detected.There were 3352 differentially expressed lncRNAs in blood samples of acute ischemic stroke patients. The validation results were consistent with gene chip data.GO and KEGG results showed these lncRNAs were mainly related to oxygen and glucose metabolism, leukocyte transendothelial migration,mitophagy and cellular senescence.Among these, lncRNA ENST00000530525 was the highly down-regulated lncRNA and mapped within the ischemic stroke associated gene anoctamin-1 (ANO1). We furtherly found lncRNA ENST00000530525 was down-regulated in hCMEC/D3 cells under 4h OGD and 20h reoxygenation(OGD4/R20) conditions. Up-regulating lncRNA ENST00000530525 decreased the cell viability while increased ANO1 expression and contributed to BBB injury of hCMEC/D3 cells after OGD4/R20.The lncRNA ENST00000530525 might plays deleterious roles in post-stroke pathogenesis. The results show light on some differentially expressed lncRNAs in human certainly participate through characteristic roles in post-stroke pathogenesis, thus, the roles and significances of some novel lncRNAs in ischemic stroke thereby warranting further study.