The Novel microRNA Rno-miR-686-3p Is Associated with the Ischaemic Penumbra.

INTRODUCTION: We explored microRNA (miRNA) profiles correlated with the penumbra in three different phases of ischaemic stroke, using a permanent middle cerebral artery occlusion (p-MCAO) rat model. MATERIALS AND METHODS: A 2-mm coronal section was cut from the optic chiasma in the caudal direction, and the penumbra was located in the area between a longitudinal line approximately 2 mm from the midline and a transverse diagonal line at the "2-o'clock" position. Total RNA was extracted from tissue specimens and peripheral blood samples, followed by deep sequencing analysis. RESULTS: We identified nine novel miRNA candidates in tissues and evaluated their expression levels using real-time quantitative polymerase chain reaction. In situ hybridization was conducted to assess miRNA localization in the brain. Of these nine candidates, we identified and characterized a novel miRNA, rno-miR-686-3p, which was localized in cell nuclei of the cortex, and associated with the penumbra. rno-miR-686-3p was downregulated at 1 (p = 0.042), 3 (p = 0.032), and 4 h (p = 0.007) post-p-MCAO in the penumbra. A total of 297 potential target genes were predicted. Moreover, functional annotation clustering and pathway enrichment analysis predicted that rno-miR-686-3p participates in transcriptional regulation and the Wnt and cyclic adenosine monophosphate (cAMP) signalling pathways. CONCLUSION: rno-miR-686-3p is a novel miRNA associated with the ischaemic penumbra that is implicated in transcriptional regulation and modulation of the Wnt and cAMP signalling pathways. Furthermore, it may serve as a possible new biomarker with potential value for detecting the existence of the penumbra.

miR-146a induces apoptosis in neuroblastoma cells by targeting BCL11A.

Aberrant expression of miR-146a has been reported to be involved in the progression and metastasis of various types of human cancers; however, its potential role in human neuroblastoma is still poorly understood. The purpose of our study was to investigate the molecular mechanism and possible role of miR-146a in human neuroblastoma. In this study, targeted genes were predicted by bioinformatic analysis and confirmed by dual-Luciferase reporter assay. The expression level of miR-146a in the human neuroblastoma SK-N-SH cell line was detected by quantitative RT-PCR. We used flow cytometric analysis to determine apoptosis and necrosis of SK-N-SH cells after transfection with miR-146a inhibitor, miR-146a mimic, and negative controls. The expression level of target genes was detected by RT-PCR and Western blotting. We identified BCL11A as a target of miR-146a. Thus, miR-146a targets the 3'UTR of BCL11A and inhibits its mRNA and protein expression. Overexpression of miR-146a can inhibit the growth and promote the apoptosis of human neuroblastoma SK-N-SH cells through inhibiting the expression of BCL11A. Furthermore, we found that upregulation of BCL11A by miR-146a inhibitor can promote SK-N-SH cells growth and protect SK-N-SH cells against apoptosis. Our results showed that miR-146a is a potential tumor suppressor gene in human neuroblastoma via directly targeting BCL11A. These findings suggest that miR-146a might be a new candidate target for treatment of human neuroblastoma.

Diagnosis of Hyperacute and Acute Ischaemic Stroke: The Potential Utility of Exosomal MicroRNA-21-5p and MicroRNA-30a-5p.

BACKGROUND: Early and accurate diagnosis of ischaemic stroke (IS) requires the use of an optimized biomarker. Exosomal microRNAs have the potential to serve as biomarkers owing to their stability and specificity. We investigated the expression levels of plasma-derived exosomal microRNA-21-5p and microRNA-30a-5p in the different phases of IS. METHODS: One hundred forty-three patients with IS and 24 non-stroke controls were enrolled. The patients were divided into the following 5 groups: 1 group for the hyperacute phase IS (HIS, within 6 h); two for the acute phase IS (AIS, including days 1-3 and days 4-7); one for the subacute phase IS (SIS, days 8-14); and one for the recovery phase IS (RIS, days >14). Plasma exosomes were isolated using a QIAGEN exoRNeasy kit and examined by transmission electron -microscopy, nanoparticle tracking, and flow cytometry. The expression levels of miRNA-21-5p and miRNA-30a-5p were detected by quantitative real-time polymerase chain reaction. RESULTS: The plasma exosomal miR-21-5p levels in SIS and RIS were significantly higher than that in controls (p < 0.05 and p < 0.01 respectively). The levels of miR-30a-5p in HIS were significantly higher (p < 0.05) and in AIS (days 1-3) were lower than that in controls (p < 0.05). In AIS (days 1-3), both miRNAs were decreased compared with the HIS group (p = 0.053 and 0.001, respectively). The area under the curve (AUC) of the miR-21-5p was 0.714 for SIS (95% CI 0.570-0.859, p = 0.007), 0.734 for RIS (95% CI 0.596-0.871, p = 0.003); the AUC of the miR-30a-5p was 0.826 for HIS (95% CI 0.665-0.988, p = 0.001), 0.438 for AIS (days 1-3; 95% CI 0.240-0.635, p = 0.516). CONCLUSIONS: The plasma-derived exosomal miR-21-5p and miRNA-30a-5p in combination are promising biomarkers for diagnosing IS and distinguishing among HIS, SIS, and RIS, especially miRNA-30a-5p for the diagnosis of the HIS phase. Our results provide a new reference for clinicians to apply in early-stage diagnosis and identifies the possible value of biomarkers for IS thrombolysis therapy.

Plasma Exosomal miRNA-122-5p and miR-300-3p as Potential Markers for Transient Ischaemic Attack in Rats.

Background: Differentiation of transient ischaemic attack (TIA) from ischaemic stroke within the thrombolysis time window is difficult. Although TIA may be diagnosed within this window, the latest imaging technologies are complex and costly. Serum markers, which are non-invasive, rapid and economic, are used for diagnosis and prognosis of various diseases. Exosome-derived miRNA markers for TIA are unknown. Methods: We examined focal brain ischaemia produced by occlusion of the middle cerebral artery (MCAo) for 5 min, 10 min, and 2 h in rats. Exosomal miRNAs with consistent trends in cerebrospinal fluid (CSF) and plasma were identified by deep sequencing and quantitative real-time polymerase chain reaction (qRT-PCR). The areas under the curve (AUC) of the receiver operating characteristic (ROC) curve were used to evaluate the diagnostic accuracy of these miRNAs for TIA in rats. Results: Rno-miR-122-5p and rno-miR-300-3p were selected. Plasma exosomal rno-miR-122-5p was significantly downregulated in 10 min ischaemic rats compared with control and 5 min plasma. Plasma exosomal rno-miR-300-3p was significantly upregulated in 5 min ischaemic rats compared with control, 10 min and 2 h rats. Plasma and CSF levels of these miRNAs were correlated. ROC analysis showed high AUC values for rno-miR-122-5p (0.960) and rno-miR-300-3p (0.970) in the 10 and 5 min rats, respectively, compared with controls. Conclusions: Plasma exosomal rno-miR-122-5p and rno-miR-300-3p may be blood-based TIA biomarkers.

Plasma Exosomal miR-422a and miR-125b-2-3p Serve as Biomarkers for Ischemic Stroke.

BACKGROUND: MircroRNA (MiRNA) levels are associated with disease pathophysiology and are high in plasma exosomes. Plasma exosomal miRNAs serve as potential therapeutic targets and diagnosis biomarkers in some diseases but few studies have examined them in Ischemic Stroke (IS). Therefore, we explored the potential predictive value of plasma exosomal miR-422a and miR-125b-2-3p in different IS phases (acute and subacute phases). METHODS: Fifty-five IS patients and 25 age and sex matched healthy controls were recruited. Patients were classified into two groups: 27 patients in acute phase (days 1-3) and 28 patients in subacute phase (days 4-14). The plasma exosomal levels of miR-422a and miR-125b-2-3p were examined via quantitative real-time polymerase chain reaction (qRT-PCR). The Areas Under the Curve (AUC) of the Receiver Operating Characteristic (ROC) curve were constructed to evaluate the diagnostic accuracy of these miRNAs in IS. RESULTS: The expression levels of plasma exosomal miR-422a and miR-125b-2-3p were significantly decreased in the subacute phase group (P<0.001, P<0.001, respectively), and the miR-422a levels were increased in the acute phase group (P<0.005) as compared to the controls. Additionally, the expression levels of plasma exosomal miR-422a and miR-125b-2-3p were significantly decreased in the subacute phase group than in the acute phase group (P<0.001, P<0.005, respectively). ROC analysis showed high AUC values for miR-422a and miR-125b-2-3p in the subacute phase group as compared to those in healthy controls: 0.971 and 0.889, respectively, and miR-422a in the acute phase group as compared to healthy controls were 0.769. CONCLUSION: Plasma exosomal miR-422a and miR-125b-2-3p may serve as blood-based biomarkers for monitoring and diagnosing in IS patients, with plasma exosomal miR-422a showing the best diagnostic value. The use of these two plasma exosomal miRNAs in combination may be powerful for determining IS stage.

MicroRNA involvement in mechanism of endogenous protection induced by fastigial nucleus stimulation based on deep sequencing and bioinformatics.

BACKGROUND: Neurogenic neuroprotection is a promising approach for treating patients with ischemic brain lesions. Fastigial nucleus stimulation (FNS) has been shown to reduce the tissue damage resulting from focal cerebral ischemia in the earlier studies. However, the mechanisms of neuroprotection induced by FNS remain unclear. MicroRNAs (miRNAs) are a newly discovered group of non-coding small RNA molecules that negatively regulate target gene expression and involved in the regulation of pathological process. To date, there is a lack of knowledge on the expression of miRNA in response to FNS. Thus, we study the regulation of miRNAs in the rat ischemic brain by the neuroprotection effect of FNS. METHODS: In this study, we used an established focal cerebral ischemia/reperfusion (IR) model in rats. MiRNA expression profile of rat ischemic cortex after 1 h of FNS were investigated using deep sequencing. Microarray was performed to study the expression pattern of miRNAs. Functional annotation on the miRNA was carried out by bioinformatics analysis. RESULTS: Two thousand four hundred ninety three miRNAs were detected and found to be miRNAs or miRNA candidates using deep sequencing technology. We found that the FNS-related miRNAs were differentially expressed according microarray data. Bioinformatics analysis indicated that several differentially expressed miRNAs might be a central node of neuroprotection-associated genetic networks and contribute to neuroprotection induced by FNS. CONCLUSIONS: MiRNA acts as a novel regulator and contributes to FNS-induced neuroprotection. Our study provides a better understanding of neuroprotection induced by FNS.

Fastigial nucleus stimulation regulates neuroprotection via induction of a novel microRNA, rno-miR-676-1, in middle cerebral artery occlusion rats.

Previous studies have shown that fastigial nucleus stimulation (FNS) reduces tissue damage resulting from focal cerebral ischemia. Although the mechanisms of neuroprotection induced by FNS are not entirely understood, important data have been presented in the past two decades. MicroRNAs (miRNAs) are a newly discovered group of non-coding small RNA molecules that negatively regulate target gene expression and are involved in the regulation of cell proliferation and cell apoptosis. To date, no studies have demonstrated whether miRNAs can serve as mediators of the brain's response to FNS, which leads to endogenous neuroprotection. Therefore, this study investigated the profiles of FNS-mediated miRNAs. Using a combination of deep sequencing and microarray with computational analysis, we identified a novel miRNA in the rat ischemic cortex after 1 h of FNS. This novel miRNA (PC-3p-3469_406), herein referred to as rno-miR-676-1, was upregulated in rats with cerebral ischemia after FNS. In vivo observations indicate that this novel miRNA may have antiapoptotic effects and contribute to neuroprotection induced by FNS. Our study provides a better understanding of neuroprotection induced by FNS. MicroRNA (miRNA) is defined as a small non-coding RNA that fulfills both the expression and biogenesis criteria. Here, we describe a novel miRNA in the rat ischemic cortex expressed after 1 h of fastigial nucleus stimulation (FNS). The miRNA was functionally characterized by secondary structure, quantitative expression, the conservation analysis, target gene analysis, and biological functions. We consider rno-miR-676-1 to be a true microRNA and present evidence for its neuroprotective effects exerted after induction by FNS.

MicroRNA-29c Correlates with Neuroprotection Induced by FNS by Targeting Both Birc2 and Bak1 in Rat Brain after Stroke.

AIMS: Studies showed fastigial nucleus stimulation (FNS) reduced brain damage, but the mechanisms of neuroprotection induced by FNS were not entirely understood; MicroRNAs are noncoding RNA molecules that regulate gene expression in a posttranscriptional manner, but their functional consequence in response to ischemia-reperfusion (IR) remains unknown. We investigated the role of microRNA-29c in the neuroprotection induced by FNS in rat. METHODS: The IR rat models were conducted 1 day after FNS. Besides, miR-29c antagomir (or agomir or control) was infused to the left intracerebroventricular 1 day before IR models were conducted. We detected differential expression of Birc2 mRNA (also Bak1mRNA and miR-29c) level among different groups by RT-qPCR. The differential expression of Birc2 protein (also Bak1 protein) level among different groups was surveyed via Western blot. The neuroprotective effects were assessed by infarct volume, neurological deficit, and apoptosis. RESULTS: MiR-29c was decreased after FNS. Moreover, miR-29c directly bound to the predicted 3'-UTR target sites of Birc2 and Bak1 genes. Furthermore, over-expression of miR-29c effectively reduced Birc2 (also Bak1) mRNA and protein levels, increased infarct volume and apoptosis, and deteriorated neurological outcomes, whereas down-regulation played a neuroprotective role. CONCLUSIONS: MiR-29c correlates with the neuroprotection induced by FNS by negatively regulating Birc2 and Bak1.

[Effects of cerebellar fastigial nucleus electrical stimulation on telomerase reverse transcriptase expression and mitochondrial apoptotic pathway in rats with focal cerebral ischemia and reperfusion].

OBJECTIVE: To study the effects of cerebellar fastigial nucleus (FN) electrical stimulation on telomerase reverse transcriptase expression and mitochondrial apoptotic pathway in rats with focal cerebral ischemia and reperfusion. METHODS: A total of 100 adult male Wistar rats were randomly divided into 3 groups: sham operation group, modeling group (2-hour cerebral ischemia, followed by 24, 48 & 72-hour reperfusion) and FN-stimulating group (electrical stimulation of FN for 1-hour one day before 2-hour cerebral ischemia, followed by 24, 48 & 72-hour reperfusion). HE (hematoxylin and eosin) and TTC (triphenyl tetrazolium chloride) staining were used to observe the morphological changes in rat brain and measure the ischemic lesion volumes. The expressions of TERT (telomerase reverse transcriptase) and Bax were detected by immunohistochemical methods and apoptotic cells by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling). The co-expression of TERT and Bax was detected by immunofluorescence double-labeling plus laser confocal microscopy. RESULTS: The morphological changes in rat brain were less greater in the FN-stimulating group than those in the modeling group. And the size of the cerebral infarct was significantly smaller in the FN-stimulating group (78.1 ± 2.9, 83.1 ± 4.5, 83.7 ± 4.8) than that in the modeling group (120.9 ± 8.2, 137.0 ± 4.2, 141.1 ± 3.3) (P < 0.05) at all reperfusion time points. As compared with the modeling group (16.1 ± 2.7, 16.9 ± 2.4, 11.6 ± 3.5), the FN-stimulating group (31.1 ± 3.5, 30.0 ± 3.4, 18.9 ± 3.3) had a significantly larger number of TERT-positive cells (P < 0.05) and a significantly reduced number of TUNEL-positive cells (49.6 ± 2.8, 67.0 ± 3.7, 46.8 ± 3.2 vs 40.2 ± 3.1, 54.8 ± 2.8, 37.3 ± 2.4) (P < 0.05). The number of Bax-positive cells at different reperfusion time points in the FN-stimulating group was not significantly different from those in the modeling group (P > 0.05). TERT partially co-localized with Bax in the cytoplasm. The number of double-labeled cells was significantly higher in the FN-stimulating group than that in the modeling group (14.1 ± 1.3, 12.9 ± 2.4, 9.0 ± 2.0 vs 8.2 ± 1.1, 6.3 ± 2.4, 6.0 ± 2.9) (P < 0.05). CONCLUSION: The expression of TERT significantly increases after a stimulation of FN. TERT may bind to Bax and inhibit Bax-mediated apoptosis by suppressing the mitochondrial relocalization of Bax from cytosol.

[Relationship between Q192R polymorphisms in paraoxonase 1 gene and young ischemic stroke].

OBJECTIVE: To investigate the relationship between polymorphisms in paraoxonase1 (PON1) gene Gln192Arg (Q192R) and arterial ischemic stroke in young adults. METHODS: The Q192R genotype was analyzed by polymerase chain reaction in 131 young adults with ischemic stroke and 135 age- and gender-matched controls. The plasma lipids were also determined in patients and controls respectively. Furthermore, carotid artery intima-media thickness (IMT) in patients were measured by carotid ultrasonography. RESULTS: The distributions of Q192R genotype frequency were significantly different between patients with ischemic stroke and control individuals. And the patients had more RR genotypes than control individuals (P < 0.05). Odds ratio (OR) for stroke were 1.743 (95% confidence interval [CI], 1.032-2.943) in subjects with RR genotype. We also studied the relationship between the polymorphisms and the lipid concentration in patients and control individuals. However, no significant association was detected between Q/R192 genotype and any of lipid measurements. Further, the prevalence of cigarette smoking, hypertension and diabetes showed no significant difference between RR and non-RR genotypes in patients. Body mass index (BMI) in two groups did not differ significantly. But IMT of patients with RR genotype obviously increased in comparison to those without RR genotype (P < 0.05). CONCLUSION: The PON1 gene Q192R polymorphism may be associated with the susceptibility of ischemic stroke in young adults. RR genotype is a genetic risk for young adults with ischemic stroke through an increased carotid artery intima-media thickness and an accelerated atherosclerotic process.