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Analysis of human stroke brain tissue resected during decompressive craniectomy or stroke-ectomy surgery

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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE155257 | GEO | 2020/07/29

REPOSITORIES: GEO

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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: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.

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Analysis of human stroke brain tissue resected during decompressive craniectomy or stroke-ectomy surgery

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