Small ncRNA expression-profiling of blood from Hemophilia A patients identifies miR-1246 as a potential regulator of Factor 8 gene
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ABSTRACT:
ORGANISM(S): synthetic construct Homo sapiens
PROVIDER: GSE65581 | GEO | 2015/02/04
SECONDARY ACCESSION(S): PRJNA274420
REPOSITORIES: GEO
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Project description:Hemophilia A (HA) is a bleeding disorder caused by deficiency of functional plasma clotting factor VIII (FVIII). In addition to genetic mutations in the gene encoding FVIII (F8), there is evidence that other molecular mechanisms may be involved in the pathobiology of HA. In this study, global small ncRNA expression profiling analysis of whole blood from HA patients, and controls, was performed using high-throughput ncRNA microarrays. Patients were further sub-divided into those that developed neutralizing-anti-FVIII antibodies (inhibitors) and those that did not. Selected differentially expressed ncRNAs were validated by quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) analysis. We identified several ncRNAs, and among them, hsa-miR-1246 was significantly up-regulated in HA patients. In addition, miR-1246 showed a six-fold higher expression in HA patients without inhibitors. We have identified an miR-1246 target site in the noncoding region of F8 mRNA and were able to confirm the suppressory role of hsa-miR-1246 on F8 expression in a stable lymphoblastoid cell line expressing FVIII. These findings suggest several testable hypotheses vis-M-CM- -vis the role of nc-RNAs in the regulation of F8 expression and the development of anti-drug antibodies to FVIII infusions used to treat HA. Whole blood samples from nine patients with hemophilia A and five controls were obtained. Among those patients with hemophilia A, three of them were patients who developed neutralizing-anti-FVIII antibodies (inhibitors) and the others are patients without FVIII inhibitors. RNA was isolated and purified from all samples using the RiboPure Blood Kit (Life Technologies) according to the manufacturerM-bM-^@M-^Ys protocol with modifications to enhance the enrichment of low molecular weight RNAs. All RNA samples were poly (A)-tailed and biotin-labeled using the FlashTag Biotin HSR RNA Labeling Kit (Affymetrix, Santa Clara, CA, USA). After labeling, the enzyme linked oligosorbent assays (ELOSA) were performed to confirm that the biotin labeling processes were successful. Biotin-labeled RNA samples (500 ng/sample) were hybridized on GeneChip miRNA 3.0 microarrays (Affymetrix). Microarray data were analyzed to identify ncRNAs that were differentially expressed in hemophilia A groups compared to controls.
2015-02-04 | E-GEOD-65581 | biostudies-arrayexpress
Project description:MicroRNAs (miRNAs) play an important role in organ development. MicroRNA expression profiling is an effective method of acquiring novel and valuable information. In this study, using miRNA microarray technology, we finally validate three over-expressed miRNAs (hsa-miR-1246, hsa-miR-1323 and hsa-miR-93) and one under-expressed miRNA (hsa-miR-143*) in NSCP group. Target gene WNT5A of hsa-miR-143* was up-regulated, while target gene FGFR2 of hsa-miR-1246, hsa-miR-1323 and hsa-miR-93 was down-regulated in the NSCP group compared to the control group. The association of miRNAs expression levels with the risk of developing NSCP, the correlation with age and gender were analyzed. The risk analysis identified that odds ratio (95% CI) of hsa-miR-1246, hsa-miR-1323 and hsa-miR-93 was higher ranged from 9.75 (1.642-57.89) to 13.67 (2.480-75.30) and pM-oM-<M-^\0.05. Neither age nor gender was associated with the four miRNAs expression levels. In male cases, the association of hsa-miR-1246, hsa-miR-1323, hsa-miR-93 and hsa-miR-143* expression levels with NSCP was observed and the p value was 0.0012, 0.0008, 0.0088 and 0.0385, respectively. Bioinformatics approaches identified nine KEGG pathways enriched in predicted target genes. In summary, we firstly defined miRNAs profiles and target genes in human soft palate tissue. Four distinctive miRNAs and their target genes expression in NSCP will advance our understanding of the genetic progress of this complex disease. Further exploration will be needed to elucidate how these miRNAs and target genes modulate signaling pathways during palatogenesis. Two-condition experiment, control (normal development people) vs. NSCP patients. Biological replicates: 6 control replicates, 10 NSCP patients replicates.
2013-06-14 | E-GEOD-47939 | biostudies-arrayexpress
Project description:Coagulation Factor VIII (FVIII) plays a pivotal role within the coagulation cascade, and deficiencies in its levels, as seen in Hemophilia A, can lead to significant health implications. Liver sinusoidal endothelial cells (LSECs) are the main producers and contributors of FVIII in blood, a fact we have previously elucidated through mRNA expression profiling when comparing these cells to other endothelial cell types. Our current investigation delves into small microRNAs, analyzing their distinct expression patterns across various endothelial cells and hepatocytes. The outcome of this exploration underscores the discernible microRNA expression differences that set LSECs apart from both hepatocytes (193 microRNAs at p < 0.05) and other endothelial cells (72 microRNAs at p < 0.05). Notably, the 134 and 35 overexpressed microRNAs in LSECs compared to hepatocytes and other endothelial cells, respectively, shed light on the unique functions of LSECs in the liver. Our investigation identified a panel of 10 microRNAs (miR-429, miR-200b-3p, miR-200a-3p, miR-216b-5p, miR-1185-5p, miR-19b-3p, miR-192-5p, miR-122-5p, miR-30c-2-3p, and miR-30a-5p) that distinctly define LSEC identity. Furthermore, our scrutiny extended to microRNAs implicated in F8 regulation, revealing a subset - miR-122-5p, miR-214-3p, miR-204-3p, and miR-2682-5p - whose expression intricately correlates with F8 expression within LSECs. This microRNA cohort emerges as a crucial modulator of F8, both directly through suppression and indirect effects on established F8-related transcription factors. The above miRNA emerged as potential targets for innovative therapies in Hemophilia A patients.
2024-02-19 | GSE242926 | GEO
Project description:MicroRNAs (miRNAs) play an important role in organ development. MicroRNA expression profiling is an effective method of acquiring novel and valuable information. In this study, using miRNA microarray technology, we finally validate three over-expressed miRNAs (hsa-miR-1246, hsa-miR-1323 and hsa-miR-93) and one under-expressed miRNA (hsa-miR-143*) in NSCP group. Target gene WNT5A of hsa-miR-143* was up-regulated, while target gene FGFR2 of hsa-miR-1246, hsa-miR-1323 and hsa-miR-93 was down-regulated in the NSCP group compared to the control group. The association of miRNAs expression levels with the risk of developing NSCP, the correlation with age and gender were analyzed. The risk analysis identified that odds ratio (95% CI) of hsa-miR-1246, hsa-miR-1323 and hsa-miR-93 was higher ranged from 9.75 (1.642-57.89) to 13.67 (2.480-75.30) and p＜0.05. Neither age nor gender was associated with the four miRNAs expression levels. In male cases, the association of hsa-miR-1246, hsa-miR-1323, hsa-miR-93 and hsa-miR-143* expression levels with NSCP was observed and the p value was 0.0012, 0.0008, 0.0088 and 0.0385, respectively. Bioinformatics approaches identified nine KEGG pathways enriched in predicted target genes. In summary, we firstly defined miRNAs profiles and target genes in human soft palate tissue. Four distinctive miRNAs and their target genes expression in NSCP will advance our understanding of the genetic progress of this complex disease. Further exploration will be needed to elucidate how these miRNAs and target genes modulate signaling pathways during palatogenesis.
2013-06-14 | GSE47939 | GEO
Project description:The human Factor VIII (F8) protein is essential for the blood coagulation cascade and specific F8 mutations cause the rare bleeding disorder Hemophilia A (HA). Here, we investigated the impact of HA-causing single-nucleotide mutations on F8 pre-mRNA splicing. We found that 14/97 (∼14.4%) coding sequence mutations tested in our study induced exon skipping. Splicing patterns of 4/11 (∼36.4%) F8 exons tested were especially sensitive to the presence of common disease-causing mutations. RNA-chemical probing analyses revealed a three-way junction structure at the 3′ end of intron 15 (TWJ-3-15). TWJ-3-15 sequesters the polypyrimidine tract, a key determinant of 3′ splice site strength. Using exon-16 of the F8 gene as a model, we designed specific antisense oligonucleotides (ASOs) that target TWJ-3-15 and identified three that promote the splicing of F8 exon-16. Interaction of TWJ-3-15 with ASOs increases accessibility of the polypyrimidine tract and inhibits the binding of hnRNPA1-dependent splicing silencing factors. Moreover, ASOs targeting TWJ-3-15 rescue diverse splicing-sensitive HA-causing mutations, most of which are distal to the 3’ splice site being impacted. The TWJ-3-15 structure and its effect on mRNA splicing provide a model for HA etiology in patients harboring specific F8 mutations and provide a framework for precision RNA-based HA therapies.
2024-01-17 | GSE230495 | GEO
Project description:Non-mutated FVIII-specific CD4 T cell epitopes have been recently found to contribute to the development of inhibitors in patients with hemophilia A (HA), while auto-reactive CD4 T cells specific to FVIII circulate in the blood of healthy individuals at a frequency close to the foreign protein Ovalbumin. Thus, although FVIII is a self-protein, the central tolerance raised against FVIII appears to be low. In this study, we conducted a comprehensive analysis of the FVIII CD4 T cell repertoire in twenty-nine healthy donors. Sequencing of the CDR3β TCR region from isolated FVIII-specific CD4 T cells revealed a limited usage and pairing of TRBV and TRBJ genes, as well as a mostly hydrophobic composition of the CDR3β region according to their auto-reactivity. FVIII repertoire is dominated by a few clonotypes, with only 13 clonotypes accounting for half of the FVIII response. Through a large-scale epitope mapping of the full-length FVIII sequence, we identified 18 immunodominant epitopes located in the A1, A3, C1, and C2 domains and covering half of the T-cell response. These epitopes exhibited a broad specificity for HLA-DR or DP molecules or both. T-cell priming with this reduced set of peptides revealed that highly expanded clonotypes specific to these epitopes were responsible individually for up to 32% of the total FVIII repertoire. These FVIII T cell epitopes and clonotypes were shared among HLA-unrelated donors tested and previously reported HA patients. Our study highlights the role of the auto-reactive T cell response against FVIII in HA and its similarity to the response observed in healthy individuals. Thus, it provides valuable insights for the development of new tolerance induction and deimmunization strategies
2024-01-22 | GSE253521 | GEO
Project description:To gain insights into the in vivo function of miRNAs in the context of periodontitis, we examined the occurrence of miRNAs in healthy and diseased gingival tissues and validated their in silico-predicted targets through mRNA profiling using whole-genome microarrays in the same specimens. Eighty-six individuals with periodontitis contributed 198 gingival papillae: 158 'diseased' (bleeding-on-probing, PD > 4 mm, and AL M-bM-^IM-% 3 mm) and 40 'healthy' (no bleeding, PD M-bM-^IM-$ 4 mm, and AL M-bM-^IM-$ 2 mm). Expression of 1,205 miRNAs was assessed by microarrays, followed by selected confirmation by quantitative RT-PCR. Predicted miRNA targets were identified and tested for enrichment by Gene Set Enrichment Analysis (GSEA). Enriched gene sets were grouped in functional categories by DAVID and Ingenuity Pathway Analysis. One hundred fifty-nine miRNAs were significantly differentially expressed between healthy and diseased gingiva. Four miRNAs (hsa-miR-451, hsa-miR-223, hsa-miR-486-5p, hsa-miR-3917) were significantly overexpressed, and 7 (hsa-miR-1246, hsa-miR-1260, hsa-miR-141, hsa-miR-1260b, hsa-miR-203, hsa-miR-210, hsa-miR-205*) were underexpressed by > 2-fold in diseased vs. healthy gingiva. GSEA and additional filtering identified 60 enriched miRNA gene sets with target genes involved in immune/inflammatory responses and tissue homeostasis. This is the first study that concurrently examined miRNA and mRNA expression in gingival tissues and will inform mechanistic experimentation to dissect the role of miRNAs in periodontal tissue homeostasis and pathology. The dataset comprise 200 gingival tissue samples from 86 patients with periodontitis. All samples are included in this series, but 2 where not included in the study, as they did not pass our QC. Each patient has one or more healthy and disease sample and samples can be of chronic or aggressive periodontitis type.
2014-02-05 | E-GEOD-54710 | biostudies-arrayexpress
Project description:Mesenchymal stem cells are known to be recruited to the tumor and contribute to a pro-inflammatory environment. The aim of this experiment was to identify potential direct targets of hsa-miR-1246 in human bone marrow derived mesenchymal stem cells in the context of inflammation. For this purpose, miR-1246 mimic or miRIDIAN microRNA Mimic Negative Control #2 both purchased from GE Healthcare Dharmacon Inc. were transiently transfected with Lipofectamine® 2000 (Invitrogen AG) at a final concentration of 30nM into primary MSCs. Transfection time was 48h according to manufacturerâs protocol. Transfections were performed in biological triplicates each.
2016-12-30 | E-MTAB-5033 | biostudies-arrayexpress
Project description:To explore the regulatory effect between miRNAs and mRNAs and the possibility of miRNAs as specific blood-based biomarkers in the developmental process of severe alopecia areata (AA), human peripheral blood samples from AA patients and healthy donors were obtained for analysis. we identified 36 significantly differentially expressed miRNAs in severe active AA patients. MiRNA target gene prediction and functional annotation analysis showed a significant enrichment in several pathways including the ribosome, cancer, cell cycle, insulin signaling, TGF-β signaling and p53 signaling pathways. Analysis of the three kinds of network showed that miR-185-5p, miR-125b-5p, and miR-186-5p might play important and synergistic roles in the developmental process of AA. According to the receiver operating characteristic curve analysis, several miRNAs were selected for the quantitative real-time PCR validation and further applied as biomarkers to classify severe active AA patients and healthy individuals. Among the miRNAs, hsa-miR-210 and hsa-miR-1246 had high prediction with high accuracy.
2018-03-01 | GSE93978 | GEO
Project description:Confirmation of the nuclease acivities at the On- and Off-target sites; Hemophilia A (HA) is caused by genetic mutations in the blood coagulation factor VIII (FVIII). Genome editing approaches can be used to target the mutated site itself in patient-derived induced pluripotent stem cells (iPSCs). However, these approaches can be hampered by difficulty preparing thousands of editing platforms for each corresponding variant found in HA patients. Here, we report a universal approach to correct the various mutations in HA patient iPSCs by the targeted insertion of the FVIII gene into the human H11 site via CRISPR/Cas9. We derived corrected clones from two types of patient iPSCs with frequencies of up to 64% and 66%, respectively, without detectable unwanted off-target mutations. Moreover, we demonstrated that endothelial cells differentiated from the corrected iPSCs successfully secreted functional protein in vitro and functionally rescued the disease phenotype in vivo. This strategy may provide a universal therapeutic method for correcting all genetic variants found in HA patients.
2019-01-05 | GSE124663 | GEO