Analysis of human stroke brain tissue resected during decompressive craniectomy or stroke-ectomy surgery
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ABSTRACT: Purpose: Signaling pathways mediated by microRNAs (miRNAs) represent one of the mechanisms that regulate stroke progression and recovery. The goal of this study is to investigate miRNA expression signatures in freshly removed human stroke brain tissue. Methods: Human brain samples (5 stroke and 3 non-stroke samples) obtained at 48-72 hours after stroke onset during craniectomy and stroke-ectomy, were subjected to histopathological and immunofluorescence microscopy analyses. NGS sequencing was performed by Qiagen company and analyses were carried out using QIAseq miRNA Quantification workflow and RNA-seq Analysis tools within CLC Genomics Workbench (version 20.0.2). Reads were normalized for expression analysis using trimmed mean of M-values method (TMM). miRNA profiling was performed using the EdgeR in Bioconductor package. Whole transcriptome RNA-sequencing Analysis: The unmapped reads from the NGS miRNA analysis were extracted, deduplicated and mapped to the genome. Gene expressions were calculated by counting number of reads mapping to the annotated gene loci. Human miR-155 Targets RT2 Profiler PCR Array (Qiagen) was performed using 3 RNA samples per stroke and control groups. The PCR Array Data analysis was performed using an automated PCR Analysis Web Portal and GeneGlobe Data Analysis (Qiagen). Results: Human stroke brain tissue was characterized by classic ischemic changes, including significant neuronal and vascular damage, and prominent edema. The absence of monocytes and notable neutrophil infiltration indicated the early stage of leukocyte response to ischemia. miRNA NGS analysis detected 36 miRNAs with significantly aberrant expression in stroke tissue, as compared to non-stroke samples. Of these miRNAs, 19 were previously identified in stroke patient blood and CSF, while dysregulation of 16 miRNAs was newly detected in this study. Bioinformatics pathway enrichment analysis demonstrated a strong association of the identified miRNAs with stroke-related biological processes and signaling pathways Conclusions: Dysregulated miRNAs detected in our study could be regarded as potential candidates for biomarkers and/or targets for therapeutic intervention. The obtained data will serve for better understanding of the molecular basis of stroke and provide valuable information for the future functional studies in the experimental models of stroke.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: The serum microvesicles of five acute ischemic stroke (AIS) and healthy controls was purified using Ribo™ Exosome Isolation Reagent (C10110-2, RIBOBIO, Guangzhou, China) and analyzed by flow cytometry and nanoparticle tracking analysis (NTA).The miRNA expression profiles of serum microvesicles of five acute ischemic stroke (AIS) and healthy controls were detected by RNA-seq using llumina HiSeqTM 2500. Results: Using an optimized data analysis workflow, 732 miRNA species were detected in total. The levels of 51 individual miRNA species were significantly different between AIS patients and healthy controls. Conclusions: Our study represents the first detailed analysis of miRNA expression profiles of serum microvesicles in AIS and healthy controls, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of miRNA content in serum microvesicles. We conclude that RNA-seq based non-coding RNA characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
Project description:Purpose: Next-generation sequencing technologies allow miRNA detection at an unprecedented sensitivity.Early- to mid-gestational fetal mammalian skin wounds heal rapidly and without scarring. The goals of this study are to obtain the differently expressed miRNAs between late- and mid-gestational fetal Keratinocytes for further studies. Methods: Keratinocytes were obtained from six fetal skin samples dividied into two groups: a mid-gestation group and a late-gestation group. RNA extracted from Keratinocytes was used to prepare a small RNA library for next-generation sequencing (NGS) using an Illumina Genome Analyzer IIx. To uncover potentially novel microRNA (miRNAs), the mirTools 2.0 web server was used to identify candidate novel human miRNAs from the NGS data.UCSC Genome Browser and the Vienna RNAfold web server were used to further validate the novel miRNA candidates. The repeatability of the expression levels detected by NGS was confirmed by real-time quantitative RT-PCR. Results: Using an optimized data analysis workflow, we detected a total of 99,354,224 raw reads from the six samples, 85,252,341 (85.81%) were high-quality reads (≥ 18nt). After alignment to the human genome (GRCH38), the number of genome-aligned reads was 74,678,115 (87.60% of the high-quality reads). The number of sequence reads that correspond to known miRNAs was 61,587,749 (82.47% of the genome-matching reads), as was determined by perfect sequence matching to the database of known miRNAs (miRBase release 18). After removing the matched non-coding RNAs (Release 10), 8,755,258 reads remained for identification of novel miRNA candidates by mirTools 2.0. The results revealed the existence of 202 novel miRNA candidates and 29 known miRNAs that were not listed in miRBase release 18. Of the 202 potential novel miRNAs, 106 candidates were detected by at least 10 counts, by NGS, indicating that they have a higher probability of being novel human miRNAs. Using Student's t-test, 173 known miRNAs and 23 novel miRNA candidates were found to be statistically significant (P values < 0.05). The expression of 22 novel miRNA candidates and 88 known miRNAs was changed by more than 2.0-fold (known: 15 up-regulated and 73 down-regulated; novel: two up-regulated and 20 down-regulated). Conclusions: Our study represents the first detailed analysis of human miRNAs in fetal Keratinocytes. Using mirTools 2.0 web server, we have revealed the existence of 202 novel miRNA candidates. Of the 202 potential novel miRNAs, 106 candidates were detected by at least 10 counts, by NGS, indicating that they have a higher probability of being novel human miRNAs. Taken together, our results provide compelling evidence that dynamic expression of miRNAs in fetal Keratinocytes at different gestational ages. MiRNAs presenting altered expression at different gestational ages in fetal Keratinocytes may contribute to scarless wound healing in early- to mid-gestational fetal Keratinocytes, and thus may be new targets for potential scar prevention and reduction therapies.
Project description:Background: Ischemic stroke is a disease with high rate of death and disability worldwide. This study investigated key circRNAs related to ischemic stroke. Methods: Three ischemic stroke patients and three healthy individuals were included in the current study to obtain the circRNA expression profiles by RNA sequencing. Through bioinformatic analysis, differentially expressed circRNAs (DEcircRNAs) were identified, and GO and pathway analyses for the host genes of DEcircRNAs were conducted. To further explore the functions of key circRNAs, a DEcircRNA-miRNA interaction network was constructed. Finally, the expression levels of selected circRNAs were validated with qRT-PCR. Results: A total of 736 DEcircRNAs were detected in ischemic stroke. Functional annotation of host genes of DEcircRNAs revealed several significantly enriched pathways, including Fc epsilon RI signaling pathway, B cell receptor signaling pathway, and T cell receptor signaling pathway. A circRNA-miRNA network, including 1544 circRNA-miRNA pairs, 456 circRNAs and 4 miRNAs, was obtained. The qRT-PCR results were largely in keeping with our RNA-seq data. Conclusion: The results of our study may help to elucidate the specific mechanism underlying ischemic stroke.
Project description:Few have characterized miRNA expression during the transition from injury to neural repair and secondary neurodegeneration following stroke in humans. We compared expression of 754 miRNAs from plasma samples collected 5, 15, and 30 days post-ischemic stroke from a discovery cohort (n=55) and 15-days post-ischemic stroke from a validation cohort (n=48) to healthy control samples (n=55 and 48 respectively) matched for age, sex, race and cardiovascular comorbidities using qRT-PCR. Eight miRNAs remained significantly altered across all time points in both cohorts including many described in acute stroke. The number of significantly dysregulated miRNAs more than doubled from post-stroke day 5 (19 miRNAs) to days 15 (50 miRNAs) and 30 (57 miRNAs). Twelve brain-enriched miRNAs were significantly altered at one or more time points (decreased expression, stroke versus controls: miR-107; increased expression: miR-99-5p, miR-127-3p, miR-128-3p, miR-181a-3p, miR-181a-5p, miR-382-5p, miR-433-3p, miR-491-5p, miR-495-3p, miR-874-3p, and miR-941). Many brain-enriched miRNAs were associated with apoptosis over the first month post-stroke whereas other miRNAs suggested a transition to synapse regulation and neuronal protection by day 30. These findings suggest that a program of decreased cellular proliferation may last at least 30 days post-stroke, and points to specific miRNAs that could contribute to neural repair in humans.
Project description:In order to determine the serum microRNAs profile from middle-old aged patients with acute ischemic stroke and investigate possible diagnostic value of these differential microRNAs.The blood samples of 117 IS patients and 82 healthy people were collected. Differential miRNAs in serum from IS and control were screened with miRNA microarray analysis, and the expression of selected miRNAs were validated by quantitative reverse-transcriptase polymerase chain reaction assays (qRT-PCR). Results: We discovered 115 differentially expressed miRNAs, among which miR-32-3p, miR-106-5p, miR-532-5p were found be related to IS for the first time. Conclusions: In the present study, we identified the changed expression pattern of miRNAs in IS. Serum miR-32-3p, miR-106-5p, miR-1246 and miR-532-5p may serve as potential diagnostic biomarkers for IS. During the initial screening stage, we divided the serum samples into five groups (10 serum samples were pooled to form a group). Group A1: A denotes thrombotic stroke and 1 denotes hepertension. Group A14: A denotes thrombotic stroke, 1 denotes hepertension and 4 denotes hyperlipidemia. Group B2: B denotes embolic stroke and 2 denotes heart disease. Group B12: B denotes embolic stroke, 1 denotes hepertension and 2 denotes heart disease. Group 0: healthy control group.
Project description:Cardiac myxoma (CM) is an important aetiology of stroke in young adults, and its diagnosis is difficult in patients having stroke because of the lack of diagnostic biomarkers. Tumour-derived exosomes play a crucial role in tumour growth, metastasis, and immune regulation, and monitor disease development. We established an RNA-sequencing dataset for long non-coding RNA (lncRNA), microRNA (miRNA), and messenger RNA (mRNA) in the plasma and tumour-derived exosomes from four patients with cardiac myxoma-related ischaemic stroke (CM-IS) and six patients with cardiac myxoma without ischaemic stroke (non-IS CM). Clean data (15.48 Gb) were obtained for lncRNAs and mRNAs. Moreover, 5,533 lncRNAs, 1,331 known miRNAs, and 412 new miRNAs were identified. Finally, gene expression profiles and differentially expressed genes were analysed in 20 samples. In the plasma samples, 74 miRNAs, 12 lncRNAs, and 693 mRNAs were identified. Tumour-derived tissue samples contained 61 miRNAs, 67 lncRNAs, and 433 mRNAs. This dataset provides a significant resource for relevant researchers to explore the potential dysregulated lncRNAs, miRNAs, and mRNAs of plasma and tumour-derived exosomes in CM-IS versus CM without stroke.
Project description:Despite the recent interest in plasma microRNA (miRNA) biomarkers in acute ischemic stroke patients, there is limited knowledge about the miRNAs directly related to stroke itself due to the multiple complications in patients, which has hindered the research progress of biomarkers and therapeutic targets of ischemic stroke. Therefore, in this study, we compared the differentially expressed miRNA profiles in the plasma of three rhesus monkeys pre- and post-cerebral ischemia. After cerebral ischemia, RFAM sequence category revealed increased ribosomic RNA (rRNA) and decreased transfer RNAs (tRNAs) in plasma. Of the 2049 miRNAs detected after cerebral ischemia, 36 were upregulated, and 76 were downregulated (fold change ≥2.0, P <0.05). For example, Mml-miR-191-5p, miR-421, miR-409-5p, and let-7g-5p were found to be significantly overexpressed, whereas mml-miR-128a-5p_R-2, miR-431_R-1, and let-7g-3p_1ss22CT were significantly downregulated. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that these differentially expressed miRNAs were implicated in the regulation of ubiquitin-mediated proteolysis and signaling pathways in cancer, glioma, chronic myeloid leukemia, and chemokine signaling. miRNA clustering analysis showed that mml-let-7g-5p and let-7g-3p_1ss22CT, which share three target genes (RB1CC1, GNG5, and CXCR4), belong to one cluster, were altered in opposite directions following ischemia. These data suggest that circulating mml-let-7g may serve as a therapeutic target for ischemic stroke.
Project description:Cardiac myxoma (CM) is an important aetiology of stroke in young adults, and its diagnosis is difficult in patients having stroke because of the lack of diagnostic biomarkers. Tumour-derived exosomes play a crucial role in tumour growth, metastasis, and immune regulation, and monitor disease development. We established an RNA-sequencing dataset for long non-coding RNA (lncRNA), microRNA (miRNA), and messenger RNA (mRNA) in the plasma and tumour-derived exosomes from four patients with cardiac myxoma-related ischaemic stroke (CM-IS) and six patients with cardiac myxoma without ischaemic stroke (non-IS CM). Clean data (15.48 Gb) were obtained for lncRNAs and mRNAs. Moreover, 5,533 lncRNAs, 1,331 known miRNAs, and 412 new miRNAs were identified. Finally, gene expression profiles and differentially expressed genes were analysed in 20 samples. In the plasma samples, 74 miRNAs, 12 lncRNAs, and 693 mRNAs were identified. Tumour-derived tissue samples contained 61 miRNAs, 67 lncRNAs, and 433 mRNAs. This dataset provides a significant resource for relevant researchers to explore the potential dysregulated lncRNAs, miRNAs, and mRNAs of plasma and tumour-derived exosomes in CM-IS versus CM without stroke.
Project description: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.
Project description:Ischemic stroke is a major public health problem that is currently among the top leading causes of serious, long-term disability worldwide. The lack of effective treatment strategies prompted a search for new molecular targets for stroke prognosis and therapy. One of the epigenetic mechanisms controlling stroke progression and recovery involves signaling pathways mediated by short non-coding RNAs called microRNAs (miRNAs). Recent studies demonstrated stroke-specific changes in miRNA expression. The main goal of the present study was identify miRNAs mediating a communication between the brain and peripheral circulation in human stroke.