Two-Tailed RT-qPCR for the Quantification of A-to-I-Edited microRNA IsoformsGjendine Voss - 2023
AbstracticroRNAs are short non-coding RNAs with important functions in the regulation of gene expression in healthy and diseased tissues. To optimally utilize
the biological and clinical information that is contained in microRNA expression levels, tools for their accurate and cost-effective quantification are needed.
While the standard method, qPCR, allows for quick and cheap microRNA
quantification, specificity is limited due to the short lengths of microRNAs and
the high similarity between closely related microRNA family members. A-toI editing can further diversify the microRNA pool by altering individual nucleotides. There is currently a lack of protocols for the accurate quantification
of A-to-I-edited microRNA isoforms using qPCR. Here, we describe a protocol to quantify microRNA editing isoforms using two-tailed RT-qPCR, with
either SYBR Green or hydrolysis probes. The user will perform reverse transcription of RNA samples, generate standard curves, and quantify the resulting
cDNA in the following qPCR step. We also give guidelines for primer design
and for the evaluation of assays using synthetic oligonucleotides. These tools
are expected to be transferable to any A-to-I-edited microRNA and its isoforms
CCR1 Mediates Müller Cell Activation and Photoreceptor Cell Death in Macular and Retinal DegenerationSarah Elbaz-Hayoun - 2023
AbstractMononuclear cells are involved in the pathogenesis of retinal diseases, including age-
related macular degeneration (AMD). Here, we examined the mechanisms that
underlie macrophage-driven retinal cell death. Monocytes were extracted from
patients with AMD and differentiated into macrophages (hMdɸs), which were
characterized based on proteomics, gene expression, and ex vivo and in vivo
properties. Using bioinformatics, we identified the signaling pathway involved in
macrophage-driven retinal cell death, and we assessed the therapeutic potential of
targeting this pathway. We found that M2a hMdɸs were associated with retinal cell
death in retinal explants and following adoptive transfer in a photic injury model.
Moreover, M2a hMdɸs express several CCRI (C-C chemokine receptor type 1)
ligands. Importantly, CCR1 was upregulated in Müller cells in models of retinal
injury and aging, and CCR1 expression was correlated with retinal damage. Lastly,
inhibiting CCR1 reduced photic-induced retinal damage, photoreceptor cell apoptosis,
and retinal inflammation. These data suggest that hMdɸs, CCR1, and Müller cells
work together to drive retinal and macular degeneration, suggesting that CCR1 may
serve as a target for treating these sight-threatening conditions.
shox2 is required for vestibular statoacoustic neuron developmentAlejandra S. Laureano - 2022
AbstractHomeobox genes act at the top of genetic hierarchies to regulate cell specification and differentiation during embryonic development. We identified the short stature homeobox domain 2 (shox2) transcription factor that is required for vestibular neuron development. shox2 transcripts are initially localized to the otic placode of the developing inner ear where neurosensory progenitors reside. To study shox2 function, we generated CRISPR-mediated mutant shox2 fish. Mutant embryos display behaviors associated with vestibular deficits and showed reduced number of anterior statoacoustic ganglion neurons that innervate the utricle, the vestibular organ in zebrafish. Moreover, a shox2-reporter fish showed labeling of developing statoacoustic ganglion neurons in the anterior macula of the otic vesicle. Single cell RNA-sequencing of cells from the developing otic vesicle of shox2 mutants revealed altered otic progenitor profiles, while single molecule in situ assays showed deregulated levels of transcripts in developing neurons. This study implicates a role for shox2 in development of vestibular but not auditory statoacoustic ganglion neurons.
MiR-214-3p targets Ras-related protein 14 (RAB14) to inhibit cellular migration and invasion in esophageal Cancer cellsPornima Phatak - 2022
AbstractBackground
MicroRNA (miR)-214-3p is emerging as an important tumor suppressor in esophageal cancer. In this study, we examined the interaction between miR-214-3p and RAB14, a membrane trafficking protein shown to exert oncogenic functions in other malignancies, in esophageal cancer cells.
Methods
Studies were performed in a human esophageal epithelial cell line and a panel of esophageal cancer cell lines, as well in human specimens. MiR-214-3p expression was measured by digital PCR. Biotinylated RNA pull-down and luciferase reporter assays assessed binding. The xCELLigence RTCA system measured cell migration and invasion in real time. A lentiviral expression vector was used to create an esophageal cancer cell line stably expressing miR-214-3p.
Results
MiR-214-3p expression was decreased in esophageal cancer cell lines and human specimens compared to non-malignant controls. RAB14 mRNA stability and protein expression were decreased following miR-214-3p overexpression. Binding between miR-214-3p and RAB14 mRNA was observed. Either forced expression of miR-214-3p or RAB14 silencing led to a marked decrease in cellular migration and invasion. Esophageal cancer cells stably expressing miR-214-3p demonstrated decreased growth in a subcutaneous murine model.
Conclusions
These results further support the tumor-suppressive role of miR-214-3p in esophageal cancer cells by demonstrating its ability to regulate RAB14 expression.
Cerebrospinal fluid of progressive multiple sclerosis patients reduces differentiation and immune functions of oligodendrocyte progenitor cellsOmri Zveik - 2022
AbstractOligodendrocyte progenitor cells (OPCs) are responsible for remyelination in the central nervous system (CNS) in health and disease. For patients with multiple sclerosis (MS), remyelination is not always successful, and the mechanisms differentiating successful from failed remyelination are not well-known. Growing evidence suggests an immune role for OPCs, in addition to their regenerative role; however, it is not clear if this helps or hinders the regenerative process. We studied the effect of cerebrospinal fluid (CSF) from relapsing MS (rMS) and progressive MS (pMS) patients on primary OPC differentiation and immune gene expression and function. We observed that CSF from either rMS or pMS patients has a differential effect on the ability of mice OPCs to differentiate into mature
oligodendrocytes and to express immune functions. CSF of pMS patients impaired differentiation into mature oligodendrocytes. In addition, it led to decreased major histocompatibility complex class (MHC)-II expression, tumor
necrosis factor (TNF)-α secretion, nuclear factor kappa-B (NFκB) activation, and less activation and proliferation of T cells. Our findings suggest that OPCs are not only responsible for remyelination, but they may also play an active role as innate immune cells in the CNS.
Inhibition of Respiratory RNA Viruses by a Composition of Ionophoric Polyphenols with Metal IonsTopaz Kreiser - 2022
AbstractControlling the infectivity of respiratory RNA viruses is critical, especially during the current SARS-CoV-2 pandemic. There is an unmet need for therapeutic agents that can reduce viral replication, preferably independent of the accumulation of viral mutations. Zinc ions have an apparent activity as modulators of intracellular viral RNA replication and thus, appear attractive in reducing viral RNA load and infectivity. However, the intracellular concentration of zinc is usually too low for achieving an optimal inhibitory effect. Various herbal polyphenols serve as excellent zinc ionophores with known antiviral properties. Here, we combined zinc picolinate with a collection of flavonoids, representing commonly used polyphenols. Copper was added to avoid ionic imbalance during treatment and to improve efficacy. Each component separately, as well as their combinations, did not interfere with the viability of cultured A549, H1299, or Vero cells in vitro as determined by MTT assay. The safe combinations were further evaluated to determine antiviral activity. Fluorescence-activated cell sorting and quantitative polymerase chain reaction were used to evaluate antiviral activity of the combinations. They revealed a remarkable (50–95%) decrease, in genome replication levels of a diverse group of respiratory RNA viruses, including the human coronavirus OC43 (HCoV-OC43; a betacoronavirus that causes the common cold), influenza A virus (IAV, strain A/Puerto Rico/8/34 H1N1), and human metapneumovirus (hMPV). Collectively, our results offer an orally bioavailable therapeutic approach that is non-toxic, naturally sourced, applicable to numerous RNA viruses, and potentially insensitive to new mutations and variants.
Investigating Interactions Between Vitis riparia Open Stomata 1 (OST1) and Inducer of CBF Expression (ICE) ProteinsMonika Jesionowska - 2022
AbstractFreezing tolerance in plants often increases due to activation of the ICE-CBF pathway. A key
activator of this pathway is Inducer of CBF Expression 1 (ICE1), which is positively regulated
by Open STomata 1 (OST1) through phosphorylation following cold stress. The phosphorylated
ICE1 activates the CBF-COR transcription cascade, resulting in gene expression associated with
increased freezing tolerance. While studied extensively in the model plant, Arabidopsis thaliana,
much is still unknown about the functions of OST1 homologues in other plant species, such as
economically important grape varieties (highly susceptible to freezing injury). Through
fluorescence microscopy and co-immunoprecipitation experiments, this thesis aimed to
investigate the OST1-ICE association from a highly frost tolerant grape, Vitis riparia. The results
of these experiments suggest that VrOST1 interacts with all VrICE proteins regardless of its
phosphorylation state and presence of the C-terminal putative interaction domain. These findings
can broaden our understanding of the Vitis CBF pathway.
Glucocorticoid-mediated induction of caveolin-1 disrupts cytoskeletal organization, inhibits cell migration and re-epithelialization of non-healing woundsIvan Jozic - 2021
AbstractAlthough impaired keratinocyte migration is a recognized hallmark of chronic wounds, the molecular mechanisms underpinning impaired cell movement are poorly understood. Here, we demonstrate that both diabetic foot ulcers (DFUs) and venous leg ulcers (VLUs) exhibit global deregulation of cytoskeletal organization in genomic comparison to normal skin and acute wounds. Interestingly, we found that DFUs and VLUs exhibited downregulation of ArhGAP35, which serves both as an inactivator of RhoA and as a glucocorticoid repressor. Since chronic wounds exhibit elevated levels of cortisol and caveolin-1 (Cav1), we posited that observed elevation of Cav1 expression may contribute to impaired actin-cytoskeletal signaling, manifesting in aberrant keratinocyte migration. We showed that Cav1 indeed antagonizes ArhGAP35, resulting in increased activation of RhoA and diminished activation of Cdc42, which can be rescued by Cav1 disruption. Furthermore, we demonstrate that both inducible keratinocyte specific Cav1 knockout mice, and MβCD treated diabetic mice, exhibit accelerated wound closure. Taken together, our findings provide a previously unreported mechanism by which Cav1-mediated cytoskeletal organization prevents wound closure in patients with chronic wounds.
Downregulation of Friend Leukemia Integration 1 (FLI1) follows the stepwise progression to gastric adenocarcinomaArmando Del Portillo - 2019
AbstractGastric adenocarcinoma (GC) is a leading cause of cancer-related deaths worldwide. The transcription factor gene Friend Leukemia Integration 1 (FLI1) is methylated and downregulated in human GC tissues. Using human GC samples, we determined which cells downregulate FLI1, when FLI1 downregulation occurs, if FLI1 downregulation correlates with clinical-pathologic characteristics, and whether FLI1 plays a role in invasion and/or proliferation of cultured cells. We analyzed stomach tissues from 98 patients [8 normal mucosa, 8 intestinal metaplasia (IM), 7 dysplasia, 91 GC] by immunohistochemistry for FLI1. Epithelial cells from normal, IM, and low-grade dysplasia (LGD) showed strong nuclear FLI1 staining. GC epithelial cells showed significantly less nuclear FLI1 staining as compared to normal epithelium, IM and LGD (P=1.2×10-5, P=1.4×10-6 and P=0.006, respectively). FLI1 expression did not correlate with tumor stage or differentiation, but was associated with patient survival, depending on tumor differentiation. We tested the functional role of FLI1 by assaying proliferation and invasion in cultured GC cells. Lentiviral-transduced FLI1 overexpression in GC AGS cells inhibited invasion by 73.5% (P = 0.001) and proliferation by 31.5% (P = 0.002), as compared to controls. Our results support a combined role for FLI1 as a suppressor of invasiveness and proliferation in gastric adenocarcinoma, specifically in the transition from pre-cancer lesions and dysplasia to invasive adenocarcinoma, and suggest that FLI1 may be a prognostic biomarker of survival in gastric cancers.
Adrenergic stimulation of adiponectin secretion in visceral mouse adipocytes is blunted in high-fat diet induced obesitySaliha Musovic - 2019
AbstractThe hormone adiponectin is secreted by white adipocytes and has been put forward as a key mediator of obesity-linked insulin resistance and the metabolic syndrome. Although adiponectin was discovered two decades ago, the knowledge about the molecular and cellular regulation of its secretion is incomplete. Here we have investigated the adrenergic regulation of adiponectin secretion in primary visceral (gonadal) adipocytes isolated from lean or obese/diabetic mice. We show that visceral adipocyte adiponectin release is triggered by cAMP/catecholamines via signalling pathways involving adrenergic beta-3-receptors (β3ARs) and Exchange Protein directly Activated by cAMP, isoform 1 (Epac1). The adrenergically stimulated adiponectin secretion is blunted in visceral adipocytes isolated from obese and diabetic mice and our results suggest the existence of a secretory defect. We have previously shown that adiponectin secretion in subcutaneous adipocytes is abolished in the obese/diabetic state due to reduced abundance of β3ARs and Epac1. However, here we show that protein levels of β3ARs and Epac1 are maintained in visceral adipocytes from obese/diabetic mice proposing that other molecular defects underlie the blunted adiponectin release. Gene expression analysis indicate diabesity-associated disturbances of the signalling downstream of Epac1 and/or the exocytotic process itself. Our study proposes that visceral adipocytes partake in the regulated secretion of adiponectin and may thus influence circulating levels of the hormone, in health and in metabolic disease.
Cellular Gene Expression during Hepatitis C Virus Replication as Revealed by Ribosome ProfilingGesche K. Gerresheim - 2019
AbstractBackground: Hepatitis C virus (HCV) infects human liver hepatocytes, often leading to liver cirrhosis and hepatocellular carcinoma (HCC). It is believed that chronic infection alters host gene expression and favors HCC development. In particular, HCV replication in Endoplasmic Reticulum (ER) derived membranes induces chronic ER stress. How HCV replication affects host mRNA translation and transcription at a genome wide level is not yet known. Methods: We used Riboseq (Ribosome Profiling) to analyze transcriptome and translatome changes in the Huh-7.5 hepatocarcinoma cell line replicating HCV for 6 days. Results: Established viral replication does not cause global changes in host gene expression—only around 30 genes are significantly differentially expressed. Upregulated genes are related to ER stress and HCV replication, and several regulated genes are known to be involved in HCC development. Some mRNAs (PPP1R15A/GADD34, DDIT3/CHOP, and TRIB3) may be subject to upstream open reading frame (uORF) mediated translation control. Transcriptional downregulation mainly affects mitochondrial respiratory chain complex core subunit genes. Conclusion: After establishing HCV replication, the lack of global changes in cellular gene expression indicates an adaptation to chronic infection, while the downregulation of mitochondrial respiratory chain genes indicates how a virus may further contribute to cancer cell-like metabolic reprogramming (“Warburg effect”) even in the hepatocellular carcinoma cells used here.
Two-tailed RT-qPCR panel for quality control of circulating microRNA studiesPeter Androvic - 2019
AbstractCirculating cell-free microRNAs are promising candidates for minimally invasive clinical biomarkers for the diagnosis, prognosis and monitoring of many human diseases. Despite substantial efforts invested in the field, the research so far has failed to deliver expected results. One of the contributing factors is general lack of agreement between various studies, partly due to the considerable technical challenges accompanying the workflow. Pre-analytical variables including sample collection, RNA isolation, and quantification are sources of bias that may hamper biological interpretation of the results. Here, we present a Two-tailed RT-qPCR panel for quality control, monitoring of technical performance, and optimization of microRNA profiling experiments from biofluid samples. The Two-tailed QC (quality control) panel is based on two sets of synthetic spike-in molecules and three endogenous microRNAs that are quantified with the highly specific Two-tailed RT-qPCR technology. The QC panel is a cost-effective way to assess quality of isolated microRNA, degree of inhibition, and erythrocyte contamination to ensure technical soundness of the obtained results. We provide assay sequences, detailed experimental protocol and guide to data interpretation. The application of the QC panel is demonstrated on the optimization of RNA isolation from biofluids with the miRNeasy Serum/Plasma Advanced Kit (Qiagen).
Metformin Increases Proliferative Activity and Viability of Multipotent Stromal Stem Cells Isolated from Adipose Tissue Derived from Horses with Equine Metabolic SyndromeAgnieszka Smieszek - 2019
AbstractIn this study, we investigated the influence of metformin (MF) on proliferation and viability of adipose-derived stromal cells isolated from horses (EqASCs). We determined the effect of metformin on cell metabolism in terms of mitochondrial metabolism and oxidative status. Our purpose was to evaluate the metformin effect on cells derived from healthy horses (EqASCHE) and individuals affected by equine metabolic syndrome (EqASCEMS). The cells were treated with 0.5 μM MF for 72 h. The proliferative activity was evaluated based on the measurement of BrdU incorporation during DNA synthesis, as well as population doubling time rate (PDT) and distribution of EqASCs in the cell cycle. The influence of metformin on EqASC viability was determined in relation to apoptosis profile, mitochondrial membrane potential, oxidative stress markers and BAX/BCL-2 mRNA ratio. Further, we were interested in possibility of metformin affecting the Wnt3a signalling pathway and, thus, we determined mRNA and protein level of WNT3A and β-catenin. Finally, using a two-tailed RT-qPCR method, we investigated the expression of miR-16-5p, miR-21-5p, miR-29a-3p, miR-140-3p and miR-145-5p. Obtained results indicate pro-proliferative and anti-apoptotic effects of metformin on EqASCs. In this study, MF significantly improved proliferation of EqASCs, which manifested in increased synthesis of DNA and lowered PDT value. Additionally, metformin improved metabolism and viability of cells, which correlated with higher mitochondrial membrane potential, reduced apoptosis and increased WNT3A/β-catenin expression. Metformin modulates the miRNA expression differently in EqASCHE and EqASCEMS. Metformin may be used as a preconditioning agent which stimulates proliferative activity and viability of EqASCs.
