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Hypoxia inducible factor-1α facilitates cellular adaptation to hypoxic conditions. Hence its tight regulation is crucial in hypoxia related diseases such as cerebral ischemia. Changes in hypoxia inducible factor-1α expression upon cerebral ischemia influence the expression of its downstream genes which eventually determines the extent of cellular damage. MicroRNAs are endogenous regulators of gene expression that have rapidly emerged as promising therapeutic targets in several diseases. In this study, we have identified miR-335 as a direct regulator of hypoxia inducible factor-1α and as a potential therapeutic target in cerebral ischemia. MiR-335 and hypoxia inducible factor-1α mRNA showed an inverse expression profile, both in vivo and in vitro ischemic conditions. Given the biphasic nature of hypoxia inducible factor-1α expression during cerebral ischemia, miR-335 mimic was found to reduce infarct volume in the early time (immediately after middle cerebral artery occlusion) of embolic stroke animal models while the miR-335 inhibitor appears to be beneficial at the late time of stroke (24 hrs after middle cerebral artery occlusion). Modulation of hypoxia inducible factor-1α expression by miR-335 also influenced the expression of crucial genes implicated in neurovascular permeability, cell death and maintenance of the blood brain barrier. These concerted effects, resulting in a reduction in infarct volume bring about a beneficial outcome in ischemic stroke.

Neuronal development is a pro-survival process that involves neurite growth, synaptogenesis, synaptic and neuronal pruning. During development, these processes can be controlled by temporal gene expression that is orchestrated by both long non-coding RNAs and microRNAs. To examine the interplay between these different components of the transcriptome during neuronal differentiation, we carried out mRNA, long non-coding RNA and microRNA expression profiling on maturing primary neurons. Subsequent gene ontology analysis revealed regulation of axonogenesis and dendritogenesis processes by these differentially expressed mRNAs and non-coding RNAs. Temporally regulated mRNAs and their associated long non-coding RNAs were significantly over-represented in proliferation and differentiation associated signalling, cell adhesion and neurotrophin signalling pathways. Verification of expression of the Axin2, Prkcb, Cntn1, Ncam1, Negr1, Nrxn1 and Sh2b3 mRNAs and their respective long non-coding RNAs in an in vitro model of ischemic-reperfusion injury showed an inverse expression profile to the maturation process, thus suggesting their role(s) in maintaining neuronal structure and function. Furthermore, we propose that expression of the cell adhesion molecules, Ncam1 and Negr1 might be tightly regulated by both long non-coding RNAs and microRNAs.

To date, miRNA expression studies on cerebral ischemia in both human and animal models have focused mainly on acute phase of ischemic stroke. In this study, we present the roles played by microRNAs in the spontaneous recovery phases in cerebral ischemia using rodent stroke models. Brain tissues were harvested at different reperfusion time points ranging from 0-168 hrs after middle cerebral artery occlusion using homologous emboli. MiRNA and mRNA expression profiles were investigated by microarray followed by multiple statistical analysis. Candidate transcripts were also validated by quantitative RT-PCR. Three specific groups of miRNAs were observed among a total of 346 differentially expressed miRNAs. miRNAs, miR-21, -142-3p, -142-5p, and -146a displayed significant upregulation during stroke recovery (48 hrs to 168 hrs) compared with those during acute phases (0 hrs to 24 hrs). On the other hand, an opposite trend was observed in the expression of miR-196a/b/c, -224 and -324-3p. Interestingly, miR-206, -290, -291a-5p and -30c-1*, positively correlated with the infarct sizes, with an initial increase up to 24hrs followed by a gradual decrease from 48 hrs to 168 hrs (R = 0.95). Taken together with the expression levels of corresponding mRNA targets, we have also found that Hedgehog, Notch, Wnt and TGF-β signaling pathways could play significant roles in stroke recovery and especially in neuronal repair.

Changes in microRNA expression have been detected in vitro in influenza infected cells, yet little is known about them in patients. microRNA profiling was performed on whole blood of H1N1 patients to identify signature microRNAs to better understand the gene regulation involved and possibly improve diagnosis. Total RNA extracted from blood samples of influenza infected patients and healthy controls were subjected to microRNA microarray. Expression profiles of circulating microRNAs were altered and distinctly different in influenza patients. Expression of highly dysregulated microRNAs were validated using quantitative PCR. Fourteen highly dysregulated miRNAs, identified from the blood of influenza infected patients, provided a clear distinction between infected and healthy individuals. Of these, expression of miR-1260, -26a, -335*, -576-3p, -628-3p and -664 were consistently dysregulated in both whole blood and H1N1 infected cells. Potential host and viral gene targets were identified and the impact of microRNA dysregulation on the host proteome was studied. Consequences of their altered expression were extrapolated to changes in the host proteome expression. These highly dysregulated microRNAs may have crucial roles in influenza pathogenesis and are potential biomarkers of influenza.

BACKGROUND: The methods currently available for diagnosis and prognosis of cerebral ischaemia still require further improvements. Micro-RNAs (small non-coding RNAs) have been recently reported as useful biomarkers in diseases such as cancer and diabetes. We therefore carried out microRNA (miRNA) profiling from peripheral blood to detect and identify characteristic patterns in ischaemic stroke. METHODS/PRINCIPAL FINDINGS: The ischaemic stroke patients aged between 18-49 years, characterized based on World Health Organization clinical criteria were further classified according to TOAST classification, a) Large-vessel atherosclerosis [n=8] b) Small-vessel disease [n=3] c) Cardioembolism [n=5] d) Undetermined cause [n=3]. The patients' functional status at the time of blood sampling (at the outpatient clinics) was evaluated with the modified Rankin Scale (mRS). Blood samples from normal (n=5) individuals were used as controls. Total RNA extracted from whole blood was subjected to miroRNA profiling and real-time PCR analysis. miRNAs that are implicated in the endothelial/vascular function, erythropoiesis, angiogenesis and neural function showed differential expression profile as compared to the normal control. Interestingly, miRNAs that are involved in hypoxic conditions have also been found in our miRNA profiles. CONCLUSION: We demonstrate that the peripheral blood miRNAs and their profiles can be developed as biomarkers in diagnosis and prognosis of cerebral ischaemic stroke. The dysregulated miRNAs have been detectable even after several months from the onset of stroke in what is usually regarded as neurologically stable patients.