scholarly journals ZBED6 regulates Igf2 expression partially through its regulation of miR483 expression

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rakan Naboulsi ◽  
Mårten Larsson ◽  
Leif Andersson ◽  
Shady Younis
Keyword(s):  
Cell Types ◽  
C2c12 Myoblast ◽  
Down Regulation ◽  
Opposite Pattern ◽  
Knock Out ◽  
Multiple Promoters ◽  
Additional Layer ◽  
Significant Enrichment ◽  
Myoblast Cells ◽  
Igf2 Expression

AbstractThe expression of Igf2 in mammals shows a complex regulation involving multiple promoters and epigenetic mechanisms. We previously identified a novel regulatory mechanism based on the interaction between the transcriptional factor ZBED6 and Igf2 intron. Disruption of the ZBED6-Igf2 interaction leads to a dramatic up-regulation of IGF2 expression postnatally. In the current study we characterize an additional layer of regulation involving miR483 encoded by another Igf2 intron. We found a highly significant up-regulation of miR483 expression when the ZBED6-Igf2 axis is disrupted in transgenic mice. Furthermore, CRISPR/Cas9 mediated knock-out of miR483 in C2C12 myoblast cells, both wild-type and cells with disrupted ZBED6-Igf2 axis (Igf2dGGCT), resulted in down-regulation of Igf2 expression and a reduced proliferation rate. This was further validated using miR483 mimics and inhibitors. RNA-seq analysis revealed a significant enrichment of genes involved in the PI3K-Akt signaling pathway among genes down-regulated in miR483−/− cells, including Igf2 down-regulation. The opposite pattern was observed in Igf2dGGCT cells, where Igf2 is up-regulated. Our data suggest a positive feedback between miR483 and Igf2 promoter activity, strongly affecting how ZBED6 controls Igf2 expression in various cell types.

scholarly journals Losing Regulation of the Extracellular Matrix is Strongly Predictive of Unfavorable Prognostic Outcome after Acute Myocardial Infarction

2020 ◽  
Vol 21 (17) ◽  
pp. 6219
Author(s):  
Pei-Hsun Sung ◽  
Kun-Chen Lin ◽  
Han-Tan Chai ◽  
John Y. Chiang ◽  
Pei-Lin Shao ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Left Ventricular ◽  
Heart Weight ◽  
Lung Injury Score ◽  
Opposite Pattern ◽  
Knock Out ◽  
Group 4 ◽  
Prognostic Outcome ◽  
Group 3

This study tested the hypothesis that MMP-9−/−tPA−/− double knock out (i.e., MTDKO) plays a crucial role in the prognostic outcome after acute myocardial infarction (AMI by ligation of left-coronary-artery) in MTDKO mouse. Animals were categorized into sham-operated controls in MTDKO animals (group 1) and in wild type (B6: group 2), AMI-MTDKO (group 3) and AMI-B6 (group 4) animals. They were euthanized, and the ischemic myocardium was harvested, by day 60 post AMI. The mortality rate was significantly higher in group 3 than in other groups and significantly higher in group 4 than in groups 1/2, but it showed no difference in the latter two groups (all p < 0.01). By day 28, the left-ventricular (LV) ejection fraction displayed an opposite pattern, whereas by day 60, the gross anatomic infarct size displayed an identical pattern of mortality among the four groups (all p < 0.001). The ratio of heart weight to tibial length and the lung injury score exhibited an identical pattern of mortality (p < 0.01). The protein expressions of apoptosis (mitochondrial-Bax/cleaved-caspase3/cleaved-PARP), fibrosis (Smad3/T-GF-ß), oxidative stress (NOX-1/NOX-2/oxidized-protein), inflammation (MMPs2,9/TNF-α/p-NF-κB), heart failure/pressure overload (BNP/ß-MHC) and mitochondrial/DNA damage (cytosolic-cytochrome-C/γ-H2AX) biomarkers displayed identical patterns, whereas the angiogenesis markers (small vessel number/CD31+cells in LV myocardium) displayed opposite patterns of mortality among the groups (all p < 0.0001). The microscopic findings of fibrotic/collagen deposition/infarct areas and inflammatory cell infiltration of LV myocardium were similar to the mortality among the four groups (all p < 0.0001). MTDKO strongly predicted unfavorable prognostic outcome after AMI.

scholarly journals Cell type resolved co-expression networks of core clock genes in brain development

2021 ◽  
Author(s):  
Surbhi Sharma ◽  
Asgar Hussain Ansari ◽  
Soundhar Ramasamy
Keyword(s):  
Circadian Clock ◽  
Single Cell ◽  
Radial Glia ◽  
Clock Genes ◽  
Neuronal Cell ◽  
Cell Types ◽  
Developing Brain ◽  
Knock Out

AbstractThe circadian clock regulates vital cellular processes by adjusting the physiology of the organism to daily changes in the environment. Rhythmic transcription of core Clock Genes (CGs) and their targets regulate these processes at the cellular level. Circadian clock disruption has been observed in people with neurodegenerative disorders like Alzheimer’s and Parkinson’s. Also, ablation of CGs during development has been shown to affect neurogenesis in both in vivo and in vitro models. Previous studies on the function of CGs in the brain have used knock-out models of a few CGs. However, a complete catalog of CGs in different cell types of the developing brain is not available and it is also tedious to obtain. Recent advancements in single-cell RNA sequencing (scRNA-seq) has revealed novel cell types and elusive dynamic cell states of the developing brain. In this study by using publicly available single-cell transcriptome datasets we systematically explored CGs-coexpressing networks (CGs-CNs) during embryonic and adult neurogenesis. Our meta-analysis reveals CGs-CNs in human embryonic radial glia, neurons and also in lesser studied non-neuronal cell types of the developing brain.

scholarly journals GSDMA drives the most replicated association with asthma in naïve CD4+ T cells

2019 ◽  
Author(s):  
Anne-Marie Madore ◽  
Lucile Pain ◽  
Anne-Marie Boucher-Lafleur ◽  
Jolyane Meloche ◽  
Andréanne Morin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
T Cells ◽  
Genetic Variants ◽  
Immune Cell ◽  
Cell Types ◽  
Sequencing Data ◽  
Opposite Pattern ◽  
Genetic Mechanisms ◽  
New Gene

AbstractBackgroundThe 17q12-21 locus is the most replicated association with asthma. However, no study had described the genetic mechanisms underlying this association considering all genes of the locus in immune cell samples isolated from asthmatic and non-asthmatic individuals.ObjectiveThis study takes benefit of samples from naïve CD4+ T cells and eosinophils isolated from the same 200 individuals to describe specific interactions between genetic variants, gene expression and DNA methylation levels for the 17q12-21 asthma locus.Methods and ResultsAfter isolation of naïve CD4+ T cells and eosinophils from blood samples, next generation sequencing was used to measure DNA methylation levels and gene expression counts. Genetic interactions were then evaluated considering genetic variants from imputed genotype data. In naïve CD4+ T cells but not eosinophils, 20 SNPs in the fourth and fifth haplotype blocks modulated both GSDMA expression and methylation levels, showing an opposite pattern of allele frequencies and expression counts in asthmatics compared to controls. Moreover, negative correlations have been measured between methylation levels of CpG sites located within the 1.5 kb region from the transcription start site of GSDMA and its expression counts.ConclusionAvailability of sequencing data from two key cell types isolated from asthmatic and non-asthmatic individuals allowed identifying a new gene in naïve CD4+ T cells that drives the association with the 17q12-21 locus, leading to a better understanding of the genetic mechanisms taking place in it.

scholarly journals Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide

2018 ◽  
Vol 115 (19) ◽  
pp. 4903-4908 ◽  
Author(s):  
Hong-Xia Wang ◽  
Ziyuan Song ◽  
Yeh-Hsing Lao ◽  
Xin Xu ◽  
Jing Gong ◽  
...  
Keyword(s):  
Gene Editing ◽  
Transfection Efficiency ◽  
Gene Activation ◽  
Cell Types ◽  
Biological Research ◽  
Knock Out ◽  
Safe Delivery ◽  
Pegylated Nanoparticles

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications.

Abstract 238: ABCA1 and HDL3 are Required to Modulate Smooth Muscle Cells Trandifferentiation in a Myocardin-miR143/145-dependent Process

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Silvia Castiglioni ◽  
Alessio Vettore ◽  
Lorenzo Arnaboldi ◽  
Laura Calabresi ◽  
Alberto Corsini ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Cell Types ◽  
Foam Cells ◽  
Knock Out ◽  
Phenotypic Changes ◽  
Basal Expression ◽  
Basal Expression Level

Cells of the artery wall may accumulate free cholesterol and cholesteryl esters becoming foam cells. Up to 50% of foam cells in human lesions originates from smooth muscle cells (SMCs). Arterial SMCs express the ATP binding cassette (ABC) transporter ABCA1 and, upon cholesterol loading, express macrophage markers and a phagocytic activity. To characterize the role of ABCA1 and HDL3 in this transdifferentiation process, we evaluated the phenotypic changes in SMCs isolated from wild type (WT) and ABCA1 knock out (KO) mice and how HDL3 affects these changes. Cholesterol loading causes the downregulation of the expression of SMC markers including ACTA2, alpha-tropomyosin and myosin heavy chain and increases the expression of macrophage-related genes such as CD68, Mac-2, SRB1, MMPs, ABCG1 and ABCA1. HDL3 treatment in WT cells is able to normalize the expression of ACTA2, while the expression of macrophage-related genes is reduced. On the contrary, the preventive effect of HDL3 is completely lost in ABCA1 KO cells. Interestingly, the presence of HDL3 does not differently affect neutral lipid accumulation in WT or ABCA1 KO cells but stimulates phospholipids removal only in WT cells. ApoAI addition does not reverse the phenotypic changes induced by cholesterol not only in KO but also in WT cells. Moreover, cholesterol loading reduces the expression of myocardin, the master SMC specific-transcriptional coactivator involved in SMC differentiation, by up to 55% (p<0.01 vs respective control) in both cell types. HDL3 normalizes myocardin levels in WT cells while it does not have any effect in ABCA1 KO cells. Similar results are obtained evaluating the levels of miR-143/145, which positively regulate myocardin. The basal expression level of KLF4, a myocardin repressor, is almost double in ABCA1 KO cells compared to WT. After cholesterol loading, KLF4 is slightly reduced in WT cells, while its expression is halved in ABCA1 KO cells. HDL3 restores KLF4 to basal levels in KO cells, but it further reduces them in WT cells. These results indicate that HDL3, modulating the miR143/145-myocardin axis in SMC, prevents the cholesterol-induced gene expression modification regardless of its cholesterol unloading capacity and the presence of ABCA1 is required.

Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates ‘reserve cells’

1998 ◽  
Vol 111 (6) ◽  
pp. 769-779 ◽  
Author(s):  
N. Yoshida ◽  
S. Yoshida ◽  
K. Koishi ◽  
K. Masuda ◽  
Y. Nabeshima
Keyword(s):  
Cell Cycle ◽  
Myogenic Differentiation ◽  
Significant Proportion ◽  
Ectopic Expression ◽  
Terminal Differentiation ◽  
Growth Conditions ◽  
Down Regulation ◽  
Undifferentiated Cells ◽  
Myoblast Cells ◽  
Myod Expression

When a proliferating myoblast culture is induced to differentiate by deprivation of serum in the medium, a significant proportion of cells escape from terminal differentiation, while the rest of the cells differentiate. Using C2C12 mouse myoblast cells, this heterogeneity observed upon differentiation was investigated with an emphasis on the myogenic regulatory factors. The differentiating part of the cell population followed a series of well-described events, including expression of myogenin, p21(WAF1), and contractile proteins, permanent withdrawal from the cell cycle and cell fusion, whereas the rest of the cells did not initiate any of these events. Interestingly, the latter cells showed an undetectable or greatly reduced level of MyoD and Myf-5 expression, which had been originally expressed in the undifferentiated proliferating myoblasts. When these undifferentiated cells were isolated and returned to the growth conditions, they progressed through the cell cycle and regained MyoD expression. These cells demonstrated identical features with the original culture on the deprivation of serum. They produced both MyoD-positive differentiating and MyoD-negative undifferentiated populations once again. Thus the undifferentiated cells in the serum-deprived culture were designated ‘reserve cells’. Upon serum deprivation, MyoD expression rapidly decreased as a result of down-regulation in approximately 50% of the cells. After this heterogenization, MyoD positive cells expressed myogenin, which is the earliest known event of terminal differentiation and marks irreversible commitment to this, while MyoD-negative cells did not differentiate and became the reserve cells. We also demonstrated that ectopic expression of MyoD converted the reserve cells to differentiating cells, indicating that down-regulation of MyoD is a causal event in the formation of reserve cells.

P4995Neuregulin-1 compensates for endothelial NO synthase deficiency in the heart and kidney

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
H Shakeri ◽  
S De Moudt ◽  
A J Leloup ◽  
G Jacobs ◽  
G R Y De Meyer ◽  
...  
Keyword(s):  
Renal Fibrosis ◽  
Cell Types ◽  
Cardiovascular Development ◽  
Wild Type ◽  
Negative Effects ◽  
Knock Out ◽  
Neuregulin 1 ◽  
Cardioprotective Effects ◽  
To Receive ◽  
Knock Out Mouse

Abstract Background Decreased eNOS activity is the hallmark of endothelial dysfunction and is associated with cardiovascular and renal disorders. Besides NO, endothelial cells produce numerous other small molecules, peptides, and proteins, which modulate the function of adjacent cells. For instance, neuregulin-1 (NRG-1) is an endothelium-derived growth factor, which plays crucial roles in cardiovascular development, has cardioprotective properties, and induces growth and differentiation of cell types in different organs, including the kidney. Purpose Although the cardioprotective effects of endothelium-derived NO and NRG-1 are well established, their interaction is not clear. Therefore, we studied the interaction between the NO/eNOS and NRG-1/ErbB signalling pathways in a transgenic eNOS knock-out mouse model (eNOS−/−) treated with subpressor doses of angiotensin II (AngII). Methods eNOS−/− mice and their wild type (WT) littermates (n=64, 15 weeks old) were randomized for implantation of osmotic minipumps with AngII (400 ng/kg.min) for 28 days or sham surgery. Mice were randomized to receive either daily NRG-1 injections (20 μg/kg, intraperitoneal) or vehicle for 4 weeks (n=8/group). Hypertrophy and fibrosis were measured in left ventricle (LV) and kidneys using echography and immunohistochemical staining. Results Although blood pressure was higher in eNOS−/− mice compared to their WT littermates, it was unaffected by a subpressor dose of AngII. Masson's trichrome staining showed that AngII significantly increased LV (interstitial and perivascular) and renal fibrosis in eNOS−/− mice, but not in WT controls (see figure for LV data). NRG-1 reversed this AngII-induced LV and renal fibrosis caused by eNOS deficiency. There was also significant hypertrophy of LV and kidneys in eNOS−/− mice treated with AngII, which was again normalized by NRG-1 treatment. Moreover, NRG-1 significantly attenuated albuminuria induced by eNOS deficiency under AngII treatment. Conclusions This study demonstrates that the anti-fibrotic and anti-hypertrophic effects of NRG-1 are independent from the NO/eNOS pathway in both heart and kidney. Strikingly, NRG-1 is able to compensate for some of the negative effects of eNOS deficiency, at least in conditions of AngII stimulation. Acknowledgement/Funding supported by university of Antwerp

scholarly journals Novel Thiosemicarbazones Sensitize Pediatric Solid Tumor Cell-Types to Conventional Chemotherapeutics through Multiple Molecular Mechanisms

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3781
Author(s):  
Silvia Paukovcekova ◽  
Jan Skoda ◽  
Jakub Neradil ◽  
Erika Mikulenkova ◽  
Petr Chlapek ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Childhood Cancer ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Metastasis Suppressor ◽  
Major Protein ◽  
Synergistic Activity ◽  
Down Regulation ◽  
Pediatric Tumor ◽  
Repair Protein

Combining low-dose chemotherapies is a strategy for designing less toxic and more potent childhood cancer treatments. We examined the effects of combining the novel thiosemicarbazones, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), or its analog, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), with the standard chemotherapies, celecoxib (CX), etoposide (ETO), or temozolomide (TMZ). These combinations were analyzed for synergism to inhibit proliferation of three pediatric tumor cell-types, namely osteosarcoma (Saos-2), medulloblastoma (Daoy) and neuroblastoma (SH-SY5Y). In terms of mechanistic dissection, this study discovered novel thiosemicarbazone targets not previously identified and which are important for considering possible drug combinations. In this case, DpC and Dp44mT caused: (1) up-regulation of a major protein target of CX, namely cyclooxygenase-2 (COX-2); (2) down-regulation of the DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT), which is known to affect TMZ resistance; (3) down-regulation of mismatch repair (MMR) proteins, MSH2 and MSH6, in Daoy and SH-SY5Y cells; and (4) down-regulation in all three cell-types of the MMR repair protein, MLH1, and also topoisomerase 2α (Topo2α), the latter of which is an ETO target. While thiosemicarbazones up-regulate the metastasis suppressor, NDRG1, in adult cancers, it is demonstrated herein for the first time that they induce NDRG1 in all three pediatric tumor cell-types, validating its role as a potential target. In fact, siRNA studies indicated that NDRG1 was responsible for MGMT down-regulation that may prevent TMZ resistance. Examining the effects of combining thiosemicarbazones with CX, ETO, or TMZ, the most promising synergism was obtained using CX. Of interest, a positive relationship was observed between NDRG1 expression of the cell-type and the synergistic activity observed in the combination of thiosemicarbazones and CX. These studies identify novel thiosemicarbazone targets relevant to childhood cancer combination chemotherapy.

scholarly journals Nckβ Adapter Controls Neuritogenesis by Maintaining the Cellular Paxillin Level

2007 ◽  
Vol 27 (17) ◽  
pp. 6001-6011 ◽  
Author(s):  
Shengxi Guan ◽  
Mei Chen ◽  
David Woodley ◽  
Wei Li
Keyword(s):  
Pc12 Cells ◽  
Cortical Neurons ◽  
State Level ◽  
Cell Types ◽  
Steady State Level ◽  
Down Regulation ◽  
Evolutionarily Conserved ◽  
Rat Cortical Neurons ◽  
Interfering Rna

ABSTRACT The SH2/SH3 adapter Nck has an evolutionarily conserved role in neurons, linking the cell surface signals to actin cytoskeleton-mediated responses. The mechanism, however, remains poorly understood. We have investigated the role of Nck/Nckα/Nck1 versus Grb4/Nckβ/Nck2 side-by-side in the process of mammalian neuritogenesis. Here we show that permanent genetic silencing of Nckβ, but not Nckα, completely blocked nerve growth factor-induced neurite outgrowth in PC12 cells and dramatically disrupted the axon and dendrite tree in primary rat cortical neurons. By screening for changes among the components reportedly present in complex with Nck, we found that the steady-state level of paxillin was significantly reduced in Nckβ knockdown, but not Nckα knockdown, neurons. Interestingly, Nckβ knockdown did not affect the paxillin level in glial cells and several other cell types of various tissue origins. Genetic silencing of paxillin blocked neuritogenesis, just like Nckβ knockdown. Reintroducing a nondegradable Nckβ into Nckβ short interfering RNA-expressing PC12 cells rescued paxillin from down-regulation and allowed the resumption of neuritogenesis. Forced expression of paxillin in Nckβ knockdown PC12 also rescued its capacity for neuritogenesis. Finally, Nckβ, but not Nckα, binds strongly to paxillin and treatment of the neurons with proteosome inhibitors prevented paxillin down-regulation in Nckβ knockdown neurons. Thus, Nckβ maintains paxillin stability during neuritogenesis.

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