Project description:This study aims to identify circulationg miR that could predict cardiovascular outcomes in patients who developed acute coronary syndrome.
Project description:Ocular Sebaceous Carcinoma (OSC) is a rare malignant tumor for which initial clinical and pathological diagnosis is often incorrect. OSC can mimic Squamous Cell Carcinoma of the Conjunctiva (SCCC). Aim of this study was to find microRNA biomarkers to distinguish OSC and SCCC from normal tissue and from each other. Clinical OSC and SCCC case files and corresponding histopathological slides were collected and reviewed. Microdissected formalin-fixed paraffin-embedded tumor and control tissue were sub-jected to semi-high throughput microRNA profiling. MicroRNA expression distinguishes OSC and SCCC from corresponding control tissues. Selected differentially expressed miRNAs were validated using single RT-PCR assays. No prognostic miRNAs could be identified that reliable predict SCCC metastasis or OSC recurrence. A com-parison between OSC (n=14) and SCCC (n=18) revealed 38 differentially expressed microRNAs (p<0.05). Differentially expressed miRNAs were selected for validation in the discovery cohort and an independent validation cohort (OSC, n=11; SCCC, n=12). At least two miRNAs miR-196b-5p (p ≤ 0.05) and miR-107 (p ≤ 0.001) displayed a statistically significant differential expression between OSC and SCCC with miR-196b-5p upregulated in SCCC and miR-107 up-regulated in OSC. In the validation cohort microRNA miR-493-3p also showed significant up-regulation in SCCC when compared to OSC (p ≤ 0.05). ROC analyses indicated that the com-bined miR-196b-5p and miR-107 expression levels predicted OSC with 90.0% sensitivity and 83.3% specificity. In conclusion, combined testing of miR-196b-5p and miR-107, can be of additional use in routine diagnostics to discriminate OSC from SCCC.
Project description:MIR-28 sibling microRNA mimics (hsa-miR-28-5p or hsa-miR-708-5p) were analyzed by the miR-eCLIP protocol established in Manakov et al. 2022, to determine direct binding targets of these microRNAs in human MCF10A breast epithelial cells.
Project description:Glioblastoma is the most prevalent and aggressive form of brain cancer. With a median overall survival of ~20 months under current standard therapy (surgery, chemotherapy, radiotherapy), novel treatment approaches are desperately needed. Immunotherapies are being investigated in clinical trials but failed so far. This includes a recent phase II clinical trial with a personalized immunotherapy based on tumor lysate-charged dendritic cells (NCT01213407). Here, we investigated tumor tissue from patients from this trial to explore glioblastoma (immuno)resistance factors and strategies to overcome them. We followed an innovative approach of combining mass spectrometry-based quantitative proteomics (n=36) with microRNA sequencing plus RT-qPCR (n=38). Protein quantification identified e.g. huntingtin interacting protein 1 (HIP1), retinol binding protein 1 (RBPP1), ferritin heavy chain (FTH1) and focal adhesion kinase 2 (FAK2) as resistance factor candidates. MicroRNA analysis identified miR-216b, miR-216a, miR-708 and let-7i as molecules potentially associated with overcoming resistance as they were enriched in patients with a comparably longer survival. In silico pathway prediction indicated they down-regulate the focal adhesion pathway – among others. FAK2 was enriched in short-term surviving immunotherapy patients. In vitro, FAK inhibitors prevented the formation of glioblastoma cell adhesion in the form of gliomaspheres. Taken together, we here mapped possible drivers of glioblastoma’s immuno)resistance in one of the largest DC vaccination tissue analysis cohorts so far – demonstrating usefulness and feasibility of combined proteomics/miRNomics approaches. Future research should investigate agents that sensitize glioblastoma to (immuno)therapy – e.g. targeting focal adhesion. Small-molecule inhibitors or microRNAs capable of altering multiple pathways at once are candidates to explore.
Project description:A long-prevailing model has held that the “seed” region (nucleotides 2-8) of a microRNA is typically sufficient to mediate target recognition and repression. However, numerous recent studies, both within the context of defining miRNA/target pairs by direct physical association and by directly assessing this model in vivo in C. elegans have brought this model into question. To test the importance of miRNA 3' pairing in vivo, in a mammalian system, we engineered a mutant murine mir-146a allele in which the 5' half of the mature microRNA retains the sequence of the wild-type mir-146a but the 3ʹ half has been altered to be anti-complementary to the wild-type miR-146a sequence. Mice homozygous or hemizygous for this mutant allele are phenotypically indistinguishable from wild-type controls and do not recapitulate any of the immunopathology previously described for mir-146a-null mice. Our results strongly support the conclusion that 3ʹ pairing is dispensable in the context of the function of a key mammalian microRNA.
Project description:Comparative proteomic study between C. elegans wild type, mir-58.1 single, mir-80; mir-58.1 double and mir-80; mir-58.1; mir-81-82 quadruple mutants.
Project description:To investigate the role and mechanism of miR-125b on NCCIT cells without bias, we analyzed differentially expression microRNA profile among miR-125b antagomir-, miR-125b agomir-, and negative control-transfected NCCIT tumor cells by microRNA-seq.
Project description:Morphine is used to sedate critically ill infants to treat painful or stressful conditions associated with intensive care. Whether neonatal morphine exposure affects microRNA (miR) expression and thereby alters mRNA regulation is unknown. We tested the hypothesis that repeated morphine treatment in stress-exposed neonatal mice alters hippocampal mRNA and miR gene expression. C57BL/6 male mice were treated from postnatal day (P) 5 to P9 with morphine at 2 or 5 mg/kg ip bid (MS5) and then exposed to stress consisting of hypoxia (100% N2 1 min and 100% O2 5 min) followed by 2h maternal separation. Control mice were untreated and dam-reared. mRNA and microRNA expression profiling was performed on hippocampal tissues at P9. Overall, MS2 and MS5 morphine treatment altered expression of a total of 150 mRNAs (>1.5 fold change, P<0.05; 36 up, 114 down), and MS5 affected 63 mRNAs. The most upregulated mRNAs were fidgetin, arginine vasopressin, and resistin-like alpha, and the most down-regulated were defensin beta 11, aquaporin 1, calmodulin-like 4, chloride intracellular channel 6, and claudin 2. Gene Set Enrichment Analysis revealed that morphine treatment affected pathways related to cell cycle, membrane function, signaling, metabolism, cell death, transcriptional regulation, and immune response. MS5 decreased expression of miR-204-5p, miR-455-3p, miR-448-3p, and miR-574-3p.Nine morphine-responsive mRNAs that are involved in neurodevelopment, neurotransmission, and inflammation are predicted targets of the aforementioned differentially expressed microRNAs These data establish that morphine produces dose-dependent changes in both hippocampal mRNA and miR gene expression in stressed neonatal mice. If permanent, morphine–mediated neuroepigenetic effects may affect long-term hippocampal function, and this provides a mechanism for the neonatal morphine-related impairment of adult learning.
Project description:A battery of spliceosome-associated proteins has been identified in microRNA (miRNA) biogenesis; however, the underlying mechanisms remain elusive. The intron lariat spliceosome (ILS) complex is highly conserved among eukaryotes and its disassembly marks the end of a canonical splicing cycle. In this study, we show that two conserved disassembly factors of the ILS complex, ILP1 and NTR1, positively regulate microRNA biogenesis through facilitating transcriptional elongation in Arabidopsis. ILP1 and NTR1 form a stable complex and co-regulate alternative splicing of more than a hundred genes across the genome including the core circadian gene LHY and some pri-miRNAs. Dysfunction in either ILP1 or NTR1 result in reduced RNA polymerase II occupancy at elongated regions of MIR chromatins, without affecting MIR promoter activity, pri-miRNA decay and DCL1 processing. Our results provide insights into the molecular mechanisms of spliceosomal machineries in non-coding RNA regulation.