ABSTRACT: Aim of the present study is to identify all circulating miRNAs that are modulated in patients with stroke, to select specific miRNAs to be used as disease biomarkers to improve both diagnosis and prognosis. Background. Stroke is the second-most common cause of death worldwide. The major factor limiting prognosis in patients affected by acute stroke is the very limited therapeutic window, so that most patients are not able receive the most successful treatments because of delays in diagnosis and to differentiate between ischemic and hemorrhagic etiology. Circulating levels of selected microRNAs (miRNAs) were found to be modulated both in animal experimental models and in patients with stroke, opening up new avenues for the identification of more effective and specific biomarkers to identify and risk-stratify stroke patients. Study aim. Aim of the present study is to identify all circulating miRNAs that are modulated in patients with stroke, to select specific miRNAs to be used as disease biomarkers to improve both diagnosis and prognosis. Methods. RNA was extracted from plasma samples using a commercial RNA extraction kit and quality of extracted material was assessed using a fluorometric electrophoretic assay (Agilent 4200 TapeStation, Santa Clara, CA, USA). MiRNA profiling was performed using the Affymetrix platform using GeneChip 4.0 (Thermo Fischer Scientific, Waltham, MA, USA). RT-PCR was performed using the Taqman protocol. MiRNA were chosen among those with the most relevant modulation between the groups. Results. Among the circulating miRNAs that were most down-regulated in stroke patients, we identified miR-3135b (20-fold, p<0.001), associated with vascular calcifications and heart failure; miR-1275 (18-fold, p=0.028), involved in cardiovascular atherosclerotic diseases and a sponge for circMAN2B2 in cancer; miR-4467 (13-fold, p=0.003), modulated in neurodegenerative diseases; and miR-7170 (7-fold, p<0.001). Among the circulating miRNAs that were most up-regulated in stroke patients, we identified miR-18a (35-fold, p=0.004), associated with stroke in the Framingham Cohort; the platelet-enriched miR-22-5p (24-fold, p=0.004), that is modulated in Huntington Disease; miR-199a (11-fold, p=0.012), a marker of brain microvascular injury and of stroke severity in rats, and miR-106b (10-fold, p=0.009), a regulator of neural stem-cell proliferation/differentiation whose level are modulated in patients with neurodegenerative diseases. Conclusions. Our results identified several circulating miRNAs that are down- of up-regulated in stroke patients. Among those with the most relevant differential expression, several miRNAs were identified that are known to play a role in the pathophysiology of neurovascular diseases, paving the way to a new class of smart pathophysiology-based biomarkers in stroke.
