The Role of MicroRNAs in Neonates with Hypoxic - Ischaemic Encephalopathy.

Abstract

With increasing knowledge on the role of microRNAs in various diseases, we aimed to identify and study the role of a candidate microRNA in neonates with hypoxic ischaemic encephalopathy (HIE). Neonates with varying degrees of encephalopathy and healthy neonates were recruited to this study. After establishing a novel technique to study microRNAs from dried blood spots, we performed discovery microRNA next generation sequencing on 32 dried blood spots from 16 neonates which identified let-7b as a potential microRNA associated with the apoptotic Hippo pathway. Validation studies using RT-qPCR on 45 neonates showed that let-7b-5p expression was increased on day 1 in neonates with moderate to severe HIE with an unfavourable outcome when compared to mild HIE. Mechanistic studies for let-7b-5p and Yes Associated Protein (YAP) in the Hippo pathway were performed on two animal models of perinatal brain injury (hypoxic-ischaemic and intrauterine inflammation models) and glucose-deprived cell cultures using fluorescence in situ hybridisation and immunohistochemistry. There was significantly reduced let-7b-5p expression in the peripheral blood of the intrauterine inflammation model, and in the cortical neurones of both animal models. Additionally, Hippo pathway activation was evident with increased neuronal/nuclear YAP ratio in the cerebral cortex of both animal models with increased apoptotic neuronal cell death, when compared to the control pups. Similar results were noted for let-7b-5p and YAP expression in glucose-deprived cell cultures. In summary, our results show that in hypoxic ischaemic stress, increased neuronal to nuclear YAP ratio with decreased neuronal let-7b-5p is linked to neuronal apoptosis. Therefore, let-7b-5p could be a potential biomarker for the severity of HIE acting through the Hippo-YAP-Let-7b axis. Further validation of this novel association of let- 7b-5p and the Hippo pathway in larger cohorts and identification of downstream targets of let-7b-5p would help to improve our understanding of the role of let-7b-5p in neonatal HIE.