RNA Synthesis by DNA Methyltransferase 1 siRNA Transfection with Cationic Liposomes into Osteoblastic Progenitor Cells Based on SiO2 Nanomagnetic Beads for Proliferation and Differentiation

2020 ◽  
Vol 20 (10) ◽  
pp. 6077-6086
Author(s):  
Qingzhen Chen ◽  
Tao Jiang ◽  
Qinshen Wang ◽  
Yongqing Huang ◽  
Min Shao
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Osteogenic Differentiation ◽  
Dna Methyltransferase ◽  
Cationic Liposomes ◽  
Precursor Cells ◽  
Dna Methyltransferase 1 ◽  
Alizarin Red ◽  
Alp Activity ◽  
Proliferation And Differentiation

DNA methylation regulated gene expression is important for osteoblast proliferation and differentiation during bone remodeling and its deregulation leads to the development of osteoporosis. DNA methyltransferase 1 (DNMT1) is an important regulator of DNA methylation. To explore the effect and mechanism of differential expression of DNMT1 in osteoblast precursor cells, DNMT1 siRNAs were designed and synthesized to interfere with DNMT1 expression in the osteoblast precursor cells, MC3T3E1 (Clone 24; MC3T3E1-24). The expression of the target gene, DNMT1, and osteogenic differentiation indicators osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting (WB). MTT assay was used to detect the effect on cell proliferation. Alkaline phosphatase (ALP) activity and alizarin red staining were used to detect the effect of DNMT1 on osteogenic differentiation. Hematoxylin and eosin (H&E) staining was used to detect the morphological changes in MC3T3E1-24 cells. Twenty-four hours following the transfection of MC3T3E1-24 cells with DNMT1 siRNA using cationic liposomes, DNMT1 mRNA and protein levels decreased significantly (P <0.001 for both). The reduced expression of DNMT1 promoted the OPG mRNA and protein expression (P <0.05), increased the ratio of OPG to RANKL (P <0.05), inhibited the expression of RANKL (P <0.01) without affecting the RANKL gene expression (not significant, P >0.05). The reduced expression of DNMT1 also promoted the proliferation of osteoblast precursor cells. In addition, ALP activity test and alizarin red staining showed that reduced expression of DNMT1 resulted in an increase in OPG/RANKL ratio and promoted the differentiation of the precursor cells. The cultured cells were found to have fibroblast-like appearance, and calcium nodules were observed after 7 days of conventional culture. In addition, to improve the efficiency of RNA extraction and save time, a type of silica nanomagnetic beads was used in the early stage of this study to extract RNA and assist qPCR detection of the target genes. The results showed that the magnetic beads could effectively extract RNA from the cells. In conclusion, low expression of DNMT1 affects proliferation and maturation of osteoblasts by upregulating OPG and OPG/RANKL ratio.

scholarly journals EGCG Prevents High Fat Diet-Induced Changes in Gut Microbiota, Decreases of DNA Strand Breaks, and Changes in Expression and DNA Methylation ofDnmt1andMLH1in C57BL/6J Male Mice

2017 ◽  
Vol 2017 ◽  
pp. 1-17 ◽  
Author(s):  
Marlene Remely ◽  
Franziska Ferk ◽  
Sonja Sterneder ◽  
Tahereh Setayesh ◽  
Sylvia Roth ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Damage ◽  
Gut Microbiota ◽  
High Fat Diet ◽  
Dna Methyltransferase ◽  
Dna Methyltransferase 1 ◽  
Male Mice ◽  
High Fat ◽  
Methyltransferase 1

Obesity as a multifactorial disorder involves low-grade inflammation, increased reactive oxygen species incidence, gut microbiota aberrations, and epigenetic consequences. Thus, prevention and therapies with epigenetic active antioxidants, (-)-Epigallocatechin-3-gallate (EGCG), are of increasing interest. DNA damage, DNA methylation and gene expression ofDNA methyltransferase 1,interleukin 6, andMutL homologue 1were analyzed in C57BL/6J male mice fed a high-fat diet (HFD) or a control diet (CD) with and without EGCG supplementation. Gut microbiota was analyzed with quantitative real-time polymerase chain reaction. An induction of DNA damage was observed, as a consequence of HFD-feeding, whereas EGCG supplementation decreased DNA damage. HFD-feeding induced a higher inflammatory status. Supplementation reversed these effects, resulting in tissue specific gene expression and methylation patterns ofDNA methyltransferase 1andMutL homologue 1. HFD feeding caused a significant lower bacterial abundance. TheFirmicutes/Bacteroidetesratio is significantly lower in HFD + EGCG but higher in CD + EGCG compared to control groups. The results demonstrate the impact of EGCG on the one hand on gut microbiota which together with dietary components affects host health. On the other hand effects may derive from antioxidative activities as well as epigenetic modifications observed on CpG methylation but also likely to include other epigenetic elements.

scholarly journals SIRT6 Promotes Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Through Activating Notch Signaling via Antagonizing DNMT1

2020 ◽  
Author(s):  
Bo Jia ◽  
Jun Chen ◽  
Qin Wang ◽  
Xiang Sun ◽  
JiuSong Han ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Dna Methyltransferases ◽  
Luciferase Reporter ◽  
Physical Interaction ◽  
Alizarin Red ◽  
Alp Activity

Abstract Background: Adipose-derived stem cells (ADSCs) are increasingly accepted as one of ideal seed cells for regenerative medicine for its potential to differentiate into multiple cell types, including osteogenic lineages. Sirtuin proteins 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase and plays important roles in a variety of biological processes, including cell differentiation.Methods: Alkaline phosphatase (ALP) activity, ALP staining, and Alizarin Red Staining was performed to explore the roles of SIRT6 in the osteogenic differentiation of ADSCs. Western blot , RT-qPCR,Luciferase reporter assay and Co-Immunoprecipitation assay were applied to confirm the relationship between of Sirt6, DNA methyltransferases (DNMTs) and NOTCHs.Results: SIRT6 leads to increased alkaline phosphatase (ALP) activity, enhanced mineralization and upregulated expression of osteogenic-related genes of human adipose-derived mesenchymal stem cells (hADSCs) in vitro and in vivo. Further mechanistic studies showed that SIRT6 regulated osteogenic differentiation of hADSCs depending on its deacetylase activity. SIRT6 selectively prevents abnormal DNA methylation of NOTCH1, NOTCH2 in hADSCs by antagonizing DNMT1. DNMT1 expression was suppressed in SIRT6 overexpression hADSCs, and knockdown partially rescued abnormal DNA methylation of NOTCH1 and NOTCH2, leading to the increased capable of osteogenic differentiation. Conculsions: SIRT6 promotes the osteogenic differentiation of hADSCs.The SIRT6 protein suppresses DNMT level via physical interaction with the DNMT1 protein, deacetylating and destabilizing DNMT1 protein,leading the activation of NOTCH1 and NOTCH2.

scholarly journals SIRT6 Promotes Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Through Activating Notch Signaling Via Antagonizing DNMT1

2020 ◽  
Author(s):  
BO JIA ◽  
Jun Chen ◽  
Qin Wang ◽  
Xiang Sun ◽  
Jiusong Han ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Dna Methyltransferases ◽  
Luciferase Reporter ◽  
Physical Interaction ◽  
Alizarin Red ◽  
Alp Activity

Abstract Background: Adipose-derived stem cells (ADSCs) are increasingly accepted as one of ideal seed cells for regenerative medicine for its potential to differentiate into multiple cell types, including osteogenic lineages. Sirtuin proteins 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase and plays important roles in a variety of biological processes, including cell differentiation.Methods: Alkaline phosphatase (ALP) activity, ALP staining, and Alizarin Red Staining was performed to explore the roles of SIRT6 in the osteogenic differentiation of ADSCs. Western blot , RT-qPCR,Luciferase reporter assay and Co-Immunoprecipitation assay were applied to confirm the relationship between of Sirt6, DNA methyltransferases (DNMTs) and NOTCHs.Results: SIRT6 leads to increased alkaline phosphatase (ALP) activity, enhanced mineralization and upregulated expression of osteogenic-related genes of human adipose-derived mesenchymal stem cells (hADSCs) in vitro and in vivo. Further mechanistic studies showed that SIRT6 regulated osteogenic differentiation of hADSCs depending on its deacetylase activity. SIRT6 selectively prevents abnormal DNA methylation of NOTCH1, NOTCH2 in hADSCs by antagonizing DNMT1. DNMT1 expression was suppressed in SIRT6 overexpression hADSCs, and knockdown partially rescued abnormal DNA methylation of NOTCH1 and NOTCH2, leading to the increased capable of osteogenic differentiation. Conculsions: SIRT6 promotes the osteogenic differentiation of hADSCs.The SIRT6 protein suppresses DNMT level via physical interaction with the DNMT1 protein, deacetylating and destabilizing DNMT1 protein,leading the activation of NOTCH1 and NOTCH2.

scholarly journals DNMT1 reads heterochromatic H4K20me3 to reinforce LINE-1 DNA methylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wendan Ren ◽  
Huitao Fan ◽  
Sara A. Grimm ◽  
Jae Jin Kim ◽  
Linhui Li ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Histone H3 ◽  
Genomic Stability ◽  
Mammalian Genome ◽  
Dna Methyltransferase 1 ◽  
Chromatin Modifications ◽  
Direct Link ◽  
Histone H4 ◽  
Radiation Induced

AbstractDNA methylation and trimethylated histone H4 Lysine 20 (H4K20me3) constitute two important heterochromatin-enriched marks that frequently cooperate in silencing repetitive elements of the mammalian genome. However, it remains elusive how these two chromatin modifications crosstalk. Here, we report that DNA methyltransferase 1 (DNMT1) specifically ‘recognizes’ H4K20me3 via its first bromo-adjacent-homology domain (DNMT1BAH1). Engagement of DNMT1BAH1-H4K20me3 ensures heterochromatin targeting of DNMT1 and DNA methylation at LINE-1 retrotransposons, and cooperates with the previously reported readout of histone H3 tail modifications (i.e., H3K9me3 and H3 ubiquitylation) by the RFTS domain to allosterically regulate DNMT1’s activity. Interplay between RFTS and BAH1 domains of DNMT1 profoundly impacts DNA methylation at both global and focal levels and genomic resistance to radiation-induced damage. Together, our study establishes a direct link between H4K20me3 and DNA methylation, providing a mechanism in which multivalent recognition of repressive histone modifications by DNMT1 ensures appropriate DNA methylation patterning and genomic stability.

Abstract 130: Hypercholesterolemia Suppresses Carotid Artery Endothelial NOS3 Expression via Epigenetic Mechanisms

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Alex Sotolongo ◽  
Yi-Zhou Jiang ◽  
John Karanian ◽  
William Pritchard ◽  
Peter Davies
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Endothelial Cells ◽  
Dna Methyltransferase ◽  
Control Group ◽  
High Cholesterol Diet ◽  
Cholesterol Diet ◽  
High Cholesterol ◽  
High Fat

Objective: One of the first clinically detectable changes in the vasculature during atherogenesis is the accumulation of cholesterol within the vessel wall. Hypercholesterolemia is characterized by dysfunctional endothelial-dependent vessel relaxation and impaired NOS3 function. Since DNA methylation at gene promoter regions strongly suppresses gene expression, we postulated that high-fat/high-cholesterol diet suppresses endothelial NOS3 through promoter DNA methylation. Methods: Domestic male pigs were fed control diet (CD) or isocaloric high fat and high cholesterol diet (HC; 12% fat and 1.5% cholesterol) for 2, 4, 8 or 12 weeks prior to tissue collection. Furthermore, to determine the effects of risk factor withdrawal, an additional group of swine received HC for 12 weeks and then CD for 8 weeks; a control group received HC continuously for 20 weeks. Endothelial cells were harvested from common carotid aorta. In parallel in vitro studies, cultured human aortic endothelial cells (HAEC) were treated with human LDL, GW3956 (LXR agonist) and RG108 (DNA methyltransferase [DNMT] inhibitor). In cells from both sources, DNA methylation at the NOS3 promoter was measured using methylation specific pyro sequencing, and endothelial gene expression was measured using RT PCR. Results: HC diet increased plasma cholesterol level from 75 mg/dl on CD to a plateau of about 540 mg/dl within 2 weeks. Endothelial NOS3 expression was significantly reduced (71±9 % of CD) after 4 weeks of HC, a level sustained at subsequent time points. Withdrawal of HC for 8 weeks did not recover NOS3 expression. After 12-week HC, the NOS3 promoter was hypermethylated. Withdrawal of HC did not reverse NOS3 promoter methylation. In vitro treatment of HAEC with human LDL (200 mg/dl total cholesterol) or GW3956 (5μM) suppressed NOS3 mRNA to 50% and 30% respectively, suggesting that LXR/RXR is involved in suppression of NOS3. Nitric oxide production was consistently suppressed by GW3959. Both could be reversed through inhibition of DNMTs by RG108. Conclusions: DNA methylation and LXR/RXR pathway can mediate the HC-suppression of endothelial NOS3. The study identifies novel pharmaceutical targets in treating endothelial dysfunction. Crosstalk between these pathways is under investigation.

Abstract 40: Disturbed Flow Alters Genomewide DNA Methylation Patterns, Regulating Endothelial Gene Expression and Atherosclerosis

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Jessilyn Dunn ◽  
Haiwei Qiu ◽  
Soyeon Kim ◽  
Daudi Jjingo ◽  
Ryan Hoffman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
Western Diet ◽  
Dependent Manner ◽  
Gene Promoters ◽  
Genome Wide ◽  
Methylation Patterns ◽  
Endothelial Gene Expression

Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow (d-flow), which alters gene expression, endothelial function, and atherosclerosis. Here, we show that d-flow regulates genome-wide DNA methylation patterns in a DNA methyltransferase (DNMT)-dependent manner. We found that d-flow induced expression of DNMT1, but not DNMT3a or DNMT3b, in mouse arterial endothelium in vivo and in cultured endothelial cells by oscillatory shear (OS) compared to unidirectional laminar shear in vitro. The DNMT inhibitor 5-Aza-2’deoxycytidine (5Aza) or DNMT1 siRNA significantly reduced OS-induced endothelial inflammation. Moreover, 5Aza reduced lesion formation in two atherosclerosis models using ApoE-/- mice (western diet for 3 months and the partial carotid ligation model with western diet for 3 weeks). To identify the 5Aza mechanisms, we conducted two genome-wide studies: reduced representation bisulfite sequencing (RRBS) and transcript microarray using endothelial-enriched gDNA and RNA, respectively, obtained from the partially-ligated left common carotid artery (LCA exposed to d-flow) and the right contralateral control (RCA exposed to s-flow) of mice treated with 5Aza or vehicle. D-flow induced DNA hypermethylation in 421 gene promoters, which was significantly prevented by 5Aza in 335 genes. Systems biological analyses using the RRBS and the transcriptome data revealed 11 mechanosensitive genes whose promoters were hypermethylated by d-flow but rescued by 5Aza treatment. Of those, five genes contain hypermethylated cAMP-response-elements in their promoters, including the transcription factors HoxA5 and Klf3. Their methylation status could serve as a mechanosensitive master switch in endothelial gene expression. Our results demonstrate that d-flow controls epigenomic DNA methylation patterns in a DNMT-dependent manner, which in turn alters endothelial gene expression and induces atherosclerosis.

miRNA-126 Inhibits Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells (BMSCs)

2022 ◽  
Vol 12 (4) ◽  
pp. 794-799
Author(s):  
Le Chang ◽  
Wei Duan ◽  
Chuang Wang ◽  
Jian Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Mrna Level ◽  
Alizarin Red ◽  
Runx2 Expression ◽  
Alp Activity ◽  
Smad4 Protein ◽  
Marrow Mesenchymal Stem Cells

This study was to determine whether microRNA (miRNA)-126 regulates osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Rat BMSCs were extracted and stimulated for osteogenic differentiation. Functional experiments were conducted to assess miR-126’s impact on BMSCs differentiation. Western blot and RT-qPCR determined miR-126 expression. ALP activity detection and alizarin red staining detection were also performed. After osteogenic differentiation of BMSCs, miR-126 expression was gradually decreased over time. Overexpression of miR-26 decreased ALP activity, Notch signaling activity as well as declined Runx2 expression and calcium Salt nodules after treatment. Importantly, we found that Smad4 serves as a target of miR-126 while upregulation of the miRNA was accompanied with the decreased Smad4 protein expression without affecting the Smad4 mRNA level. In conclusion, miR-126 restrains osteogenic differentiation through inhibition of SMAD4 signaling, providing a novel insight into the mechanism.

scholarly journals Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

2020 ◽  
Author(s):  
Daniel M. Sapozhnikov ◽  
Moshe Szyf
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Promoter Region ◽  
Dna Methyltransferase ◽  
Dna Demethylation ◽  
New Method ◽  
Inducible Promoters ◽  
Specific Targeting ◽  
Main Effect

AbstractAlthough associations between DNA methylation and gene expression were established four decades ago, the causal role of DNA methylation in gene expression remains unresolved. Different strategies to address this question were developed; however, all are confounded and fail to disentangle cause and effect. We developed here a highly effective new method using only deltaCas9(dCas9):gRNA site-specific targeting to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzymatic activity, enabling examination of the role of DNA methylation per se in living cells. We show that the extensive induction of gene expression achieved by TET/dCas9-based targeting vectors is confounded by DNA methylation-independent activities, inflating the role of DNA methylation in the promoter region. Using our new method, we show that in several inducible promoters, the main effect of DNA methylation is silencing basal promoter activity. Thus, the effect of demethylation of the promoter region in these genes is small, while induction of gene expression by different inducers is large and DNA methylation independent. In contrast, targeting demethylation to the pathologically silenced FMR1 gene targets robust induction of gene expression. We also found that standard CRISPR/Cas9 knockout generates a broad unmethylated region around the deletion, which might confound interpretation of CRISPR/Cas9 gene depletion studies. In summary, this new method could be used to reveal the true extent, nature, and diverse contribution to gene regulation of DNA methylation at different regions.

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