3D genome organization

Copyright © The Author(s) 2022. Our genomes are highly organized spatially in three-dimensions (3D). In interphase nuclei, the genome is anchored and regulated by various nuclear scaffolds and structures, including the nuclear lamina at the nuclear edge, and nucleoli located more internally within the nucleoplasm. Recently, great effort has been made to understand the intricacies of 3D genome organization and its relevance to genomic and nuclear function. Over the years, many concepts, mathematical models, visual and biochemical methods, and analysis pipelines have been presented to study various aspects of this organization in a multidisciplinary manner, such as is also reflected within this collection

Deficiency of the miR-29a/b-1 cluster leads to ataxic features and cerebellar alterations in mice

miR-29 is expressed strongly in the brain and alterations in expression have been linked to several neurological disorders. To further explore the function of this miRNA in the brain, we generated miR-29a/b-1 knockout animals. Knockout mice develop a progressive disorder characterized by locomotor impairment and ataxia. The different members of the miR-29 family are strongly expressed in neurons of the olfactory bulb, the hippocampus and in the Purkinje cells of the cerebellum. Morphological analysis showed that Purkinje cells are smaller and display less dendritic arborisation compared to their wildtype littermates. In addition, a decreased number of parallel fibers form synapses on the Purkinje cells. We identified several mRNAs significantly up-regulated in the absence of the miR-29a/b-1 cluster. At the protein level, however, the voltage-gated potassium channel Kcnc3 (Kv3.3) was significantly up-regulated in the cerebella of the miR-29a/b knockout mice. Dysregulation of KCNC3 expression may contribute to the ataxic phenotype

Imaging individual microRNAs in single mammalian cells in situ

MicroRNAs (miRNAs) are potent negative regulators of gene expression that have been implicated in most major cellular processes. Despite rapid advances in our understanding of miRNA biogenesis and mechanism, many fundamental questions still remain regarding miRNA function and their influence on cell cycle control. Considering recent reports on the impact of cell-to-cell fluctuations in gene expression on phenotypic diversity, it is likely that looking at the average miRNA expression of cell populations could result in the loss of important information connecting miRNA expression and cell function. Currently, however, there are no efficient techniques to quantify miRNA expression at the single-cell level. Here, a method is described for the detection of individual miRNA molecules in cancer cells using fluorescence in situ hybridization. The method combines the unique recognition properties of locked nucleic acid probes with enzyme-labeled fluorescence. Using this approach, individual miRNAs are identified as bright, photostable fluorescent spots. In this study, miR-15a was quantified in MDA-MB-231 and HeLa cells, while miR-155 was quantified in MCF-7 cells. The dynamic range was found to span over three orders of magnitude and the average miRNA copy number per cell was within 17.5% of measurements acquired by quantitative RT-PCR

Upregulation of miR-31* Is Negatively Associated with Recurrent/Newly Formed Oral Leukoplakia

BACKGROUND: Oral leukoplakia (OLK) is a potentially malignant disorder of the oral cavity. However, the underlying mechanism of OLK is still unclear. In this study, we explore possible miRNAs involved in OLK. METHODOLOGY/PRINCIPAL FINDINGS: Using miRNA microarrays, we profiled miRNA expression in OLK and malignantly transformed OLK (mtOLK) tissue samples. The upregulation of miR-31*, miR-142-5p, miR-33a, miR-1259, miR-146b-5p, miR-886-3p, miR-886-5p, miR-519d, and miR-301a along with the downregulation of miR-572, miR-611, miR-602, miR-675, miR-585, miR-623, miR-637, and miR-1184 in mtOLK were new observations. Fluorescence in situ hybridization (FISH) analyses confirmed that miR-31* is highly expressed in mtOLK. There was a significant difference between the FISH score (p<0.05) in patients with or without recurrent/newly formed OLK. Functional analyses demonstrated that a miR-31* inhibitor decreased apoptosis in the Leuk-1, which is an immortalized oral epithelial cell line spontaneously derived from an oral leukoplakia lesion. miR-31* regulated apoptosis, cell proliferation, migration, and invasion in the HOIEC, which is a HPV E6/E7-immortalized oral epithelial cell line. Furthermore, miR-31* modulated the biological functions of apoptosis, cell proliferation, cell cycle, migration, and invasion in the oral squamous cell carcinoma cell line, Cal-27. Using bioinformatic analyses and dual luciferase reporter assays, we determined that the 3' untranslated region of fibroblast growth factor 3 (FGF3) is the target of miR-31*. Expression of FGF3 was downregulated or upregulated in the presence of a miR-31* mimic or inhibitor, respectively. CONCLUSIONS/SIGNIFICANCE: Upregulation of miR-31* is negatively associated with recurrent/newly formed OLK. MiR-31* may exert similar but distinguishable effects on biological function in oral cells with different malignant potential. FGF3 is the target of miR-31*. miR-31* may play an important role during OLK progression through regulating FGF3. MiRNA* strands may also have prominent roles in oral carcinogenesis

High levels of microRNA-21 in the stroma of colorectal cancers predict short disease-free survival in stage II colon cancer patients

Approximately 25% of all patients with stage II colorectal cancer will experience recurrent disease and subsequently die within 5 years. MicroRNA-21 (miR-21) is upregulated in several cancer types and has been associated with survival in colon cancer. In the present study we developed a robust in situ hybridization assay using high-affinity Locked Nucleic Acid (LNA) probes that specifically detect miR-21 in formalin-fixed paraffin embedded (FFPE) tissue samples. The expression of miR-21 was analyzed by in situ hybridization on 130 stage II colon and 67 stage II rectal cancer specimens. The miR-21 signal was revealed as a blue chromogenic reaction, predominantly observed in fibroblast-like cells located in the stromal compartment of the tumors. The expression levels were measured using image analysis. The miR-21 signal was determined as the total blue area (TB), or the area fraction relative to the nuclear density (TBR) obtained using a red nuclear stain. High TBR (and TB) estimates of miR-21 expression correlated significantly with shorter disease-free survival (p = 0.004, HR = 1.28, 95% CI: 1.06–1.55) in the stage II colon cancer patient group, whereas no significant correlation with disease-free survival was observed in the stage II rectal cancer group. In multivariate analysis both TB and TBR estimates were independent of other clinical parameters (age, gender, total leukocyte count, K-RAS mutational status and MSI). We conclude that miR-21 is primarily a stromal microRNA, which when measured by image analysis identifies a subgroup of stage II colon cancer patients with short disease-free survival

Pre-microRNA and Mature microRNA in Human Mitochondria

Chantier qualité GAInternational audienceBACKGROUND: Because of the central functions of the mitochondria in providing metabolic energy and initiating apoptosis on one hand and the role that microRNA (miRNA) play in gene expression, we hypothesized that some miRNA could be present in the mitochondria for post-transcriptomic regulation by RNA interference. We intend to identify miRNA localized in the mitochondria isolated from human skeletal primary muscular cells. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the potential origin of mitochondrial miRNA, we in-silico searched for microRNA candidates in the mtDNA. Twenty five human pre-miRNA and 33 miRNA aligments (E-value35) for the smallest RNA input concentration and 204 miRNA for the maximum RNA input concentration. In silico analysis predicted 80 putative miRNA target sites in the mitochondrial genome (E-value<0.05). CONCLUSIONS/SIGNIFICANCE: The present study experimentally demonstrated for the first time the presence of pre-miRNA and miRNA in the human mitochondria isolated from skeletal muscular cells. A set of miRNA were significantly detected in mitochondria fraction. The origin of these pre-miRNA and miRNA should be further investigate to determine if they are imported from the cytosol and/or if they are partially processed in the mitochondria