Regulatory Mechanisms Underlying MiR-296-Mediated Angiogenesis After Cerebral Ischemic Injury

Background and aims:Stroke is a leading cause of death and disability worldwide, and ischemic stroke is the dominant subtype, significantly threatening the human health with a high morbidity, disability, and mortality. In the absence of effective treatment due to the short but best therapeutic window time, the restoration or improvement of reduced regional cerebral blood supply in a timely manner is very critical for improving stroke outcomes and post-stroke functional recovery. Therefore, angiogenesis has come into sharper focus in the field of ischemic stroke. Most recently, microRNAs which are20-25nucleotide non-protein-coding RNA molecules have been identified as negative regulators of gene expression in a post-transcriptional manner, modulating the translational efficiency and/or the stability of target messenger RNAs (mRNAs). Accumulating studies have demonstrated that miRNAs are essential determinants of vascular endothelial cell biology/angiogenesis as well as contributors to stroke pathogenesis. The identification of specific miRNAs as a key regulators of the response to ischemia has opened new clinical avenues. Previous research has shown that glioma cells and angiogenic growth factors elevate the level of miR-296in primary human brain microvascular endothelial cells in culture. Growth factor-induced miR-296contributes significantly to angiogenesis by directly targeting hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) mRNA, leading to decreased levels of HGS and thereby reducing HGS-mediated degradation of the growth factor receptors VEGFR2, which is one of the most powerful pro-angiogenesis factor at present, which can participate in each link of the angiogenesis directly or indirectly. Thus, it could reasonably be inferred that miR-296should take part in and/or enhance post-ischemic angiogenesis in cerebral ischemic. In this study, we show that miR-296, targeting HGS mRNA, is involved in angiogenesis after cerebral ischemia and provide potential mechanisms that miR-296may up-regulate VEGF/VEGF2signaling but down-regulate DLL4/Notch signaling for capillary formation in vitro.Materials and Results:Rat cerebral ischemic model was established by right middle cerebral artery occlusion. Animals were randomly divided into four groups (sham, Id,3d and7d), and real-time quantitative polymerase chain reaction and western blot analyses were performed to examine the expression levels of miR-296and its target-HGS respectively. Angiogenesis was assessed by the CD105marker. Our results showed that miR-296and angiogenesis was significantly up-regulated, while HGS was markedly down-regulated after ischemic injury. Adenovirus-mediated overexpression of miR-296markedly enhanced formation of capillary-like structures in human umbilical vein endothelial cells, in parallel to significantly increased expression of vascular endothelial growth factor (VEGF) and VEGF receptor2(VEGFR2) and decreased expression of DLL4and Notch1.Conclusions:This study provides in vivo and in vitro evidence suggesting that miR-296promotes angiogenesis through up-regulating VEGF/VEGFR2and down-regulating DLL4/Notchl expression after cerebral ischemic injury.