scholarly journals Role of miR-383 and miR-146b in different propensities to obesity in male mice

2017 ◽  
Vol 234 (2) ◽  
pp. 201-216 ◽  
Author(s):  
Shu-Fang Xia ◽  
Xiao-Mei Duan ◽  
Xiang-Rong Cheng ◽  
Li-Mei Chen ◽  
Yan-Jun Kang ◽  
...  
Keyword(s):  
Hepatic Steatosis ◽  
Target Genes ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Stem Loop ◽  
Next Generation Sequencing Technology ◽  
Diet Induced Obesity ◽  
Primary Mouse ◽  
Mouse Hepatocytes ◽  
Cellular Lipid Accumulation

The study was designed to investigate the possible mechanisms of hepatic microRNAs (miRs) in regulating local thyroid hormone (TH) action and ultimately different propensities to high-fat diet (HFD)-induced obesity. When obesity-prone (OP) and obesity-resistant (OR) mice were fed HFD for 7 weeks, OP mice showed apparent hepatic steatosis, with significantly higher body weight and lower hepatic TH receptor b (TRb) expression and type 1 deiodinase (DIO1) activity than OR mice. Next-generation sequencing technology revealed that 13 miRs in liver were dysregulated between the two phenotypes, of which 8 miRs were predicted to target on Dio1 or TRb. When mice were fed for 17 weeks, OR mice had mild hepatic steatosis and increased Dio1 and TRb expression than OP mice, with downregulation of T3 target genes (including Srebp1c, Acc1, Scd1 and Fasn) and upregulation of Cpt1α, Atp5c1, Cox7c and Cyp7a1. A stem-loop qRT-PCR analysis confirmed that the levels of miR-383, miR-34a and miR-146b were inversely correlated with those of DIO1 or TRb. Down-regulated expression of miR-383 or miR-146b by miR-383 inhibitor (anti-miR-383) or miR-146b inhibitor (anti-miR-146b) in free fatty acid-treated primary mouse hepatocytes led to increased DIO1 and TRb expressions, respectively, and subsequently decreased cellular lipid accumulation, while miR-34a inhibitor (anti-miR-34a) transfection had on effects on TRb expression. Luciferase reporter assay illustrated that miR-146b could directly target TRb 3′untranslated region (3′UTR). These findings suggested that miR-383 and miR-146b might play critical roles in different propensities to diet-induced obesity via targeting on Dio1 and TRb, respectively.

scholarly journals Review of: Metalloproteinase axes increase β-catenin signaling in primary mouse mammary epithelial cells lacking TIMP3

2007 ◽  
Vol 10 (8) ◽  
Author(s):  
D. S. Salomon
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Target Genes ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Specific Effects ◽  
Primary Mouse ◽  
Ductal Elongation

Citation of original article:C. V. Hojilla, I. Kim, Z. Kassiri, J. E. Fat, H. Fang, R. Khokha. Journal of Cell Science 2007; 120(6): 1050–1060.Abstract of the original article:Multiple cancers exhibit mutations in β-catenin that lead to increased stability, altered localization or amplified activity. β-Catenin is situated at the junction between the cadherin-mediated cell adhesion and Wnt signaling pathways, and TIMP3 functions to alter β-catenin signaling. Here we demonstrate that primary mouse embryonic fibroblasts (MEFs) and mammary epithelial cells (MECs) deficient in Timp3 have increased β-catenin signaling. Functionally, the loss of TIMP3 exerted cell-type-specific effects, with Timp3−/− MEFs being more sensitive and Timp3−/− MECs more resistant to EGTA-induced cell detachment than the wild type. Timp3−/− MECs had higher dephosphorylated β-catenin levels and increased β-catenin transcriptional activity as measured by TCF/LEF-responsive reporter assays. Real-time PCR analysis of β-catenin target genes in MEFs and MECs showed no alteration in Myc, decreased Ccnd1 (cyclin D1) and increased Mmp7 mRNA levels upon loss of TIMP3, with the latter occurring only in epithelial cells. Recombinant TIMP3 and synthetic metalloproteinase inhibitors reverted the increase in dephosphorylated β-catenin, decrease in Ccnd1 gene expression and increase in Mmp7 gene expression. Physiologically, Timp3−/− mammary glands displayed accelerated mammary ductal elongation during pubertal morphogenesis. Gain-of-function studies using slow-release TIMP-containing pellets revealed distinct effects of individual TIMPs on ductal morphogenesis. Recombinant TIMP1, TIMP3 and TIMP4 inhibited ductal elongation whereas TIMP2 promoted this process.

scholarly journals Elevated miR-10a-5p facilitates cell cycle and restrains adipogenic differentiation via targeting Map2k6 and Fasn, respectively

2020 ◽  
Vol 52 (11) ◽  
pp. 1227-1235
Author(s):  
Xiaoyu Wang ◽  
Huifang Zhang ◽  
Meixue Xu ◽  
Xin’E Shi ◽  
Gongshe Yang ◽  
...  
Keyword(s):  
Target Genes ◽  
Adipogenic Differentiation ◽  
Luciferase Reporter ◽  
Proliferative Stage ◽  
Primary Mouse ◽  
Direct Targeting ◽  
Treatment Of Obesity ◽  
The Stability ◽  
Oil Red O Staining

Abstract miRNAs are a small class of noncoding RNAs that perform biological functions by regulating the stability or translation of target genes in various biological processes. This study illustrated the role of miR-10a-5p, which is relatively enriched in adipose tissues, using primary mouse preadipocytes as model. With elevated miR-10a-5p expression, the proliferative ability of mouse preadipocytes was significantly enhanced, indicated by increased EdU+ cells and G1/S transition, accompanied by upregulated Cyclin B, Cyclin D and PCNA and downregulated p21 and p27. Meanwhile, the adipogenic differentiation was significantly attenuated by elevated miR-10a-5p, supported by Oil Red O staining and suppressed PPARγ and aP2 expression. Furthermore, Map2k6 and Fasn were predicted to be the target genes of miR-10a-5p in silico, and dual luciferase reporter assay confirmed the direct targeting effects. Western blot analysis results showed that miR-10a-5p specially reduced Map2k6 expression at the proliferative stage without affecting Fasn expression, while significantly restrained Fasn expression with unchanged Map2k6 expression during adipogenic differentiation. Taken together, these results revealed a potential role of miR-10a-5p in adipogenesis and in the treatment of obesity.

scholarly journals Regulation of Cytochrome P450 2a5 by Artemisia capillaris and 6,7-Dimethylesculetin in Mouse Hepatocytes

2021 ◽  
Vol 12 ◽  
Author(s):  
Sangsoo Daniel Kim ◽  
Larry Morgan ◽  
Elyse Hargreaves ◽  
Xiaoying Zhang ◽  
Zhihui Jiang ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Nuclear Translocation ◽  
Constitutive Androstane Receptor ◽  
Elevated Serum ◽  
Regulatory Control ◽  
Herbal Remedies ◽  
Luciferase Reporter ◽  
Artemisia Capillaris ◽  
Primary Mouse ◽  
Mouse Hepatocytes

Jaundice is a potentially fatal condition resulting from elevated serum bilirubin levels. For centuries, herbal remedies containing Artemisia capillaris Thunb. including the compound 6,7-dimethylesculetin (DE) have been used in Asia to prevent and treat jaundice in neonates. DE activates an important regulator of bilirubin metabolism, the constitutive androstane receptor (CAR), and increases bilirubin clearance. In addition, murine cytochrome P450 2a5 (Cyp2a5) is known to be involved in the oxidative metabolism of bilirubin. Moreover, treatment of mice with phenobarbital, a known inducer of both CAR and Cyp2a5, increases expression of Cyp2a5 suggesting a potential relationship between CAR and Cyp2a5 expression. The aim of this study is to investigate the influence of Artemisia capillaris and DE on the expression and regulatory control of Cyp2a5 and the potential involvement of CAR. Treatment of mouse hepatocytes in primary culture with DE (50 μM) significant increased Cyp2a5 mRNA and protein levels. In mice, Artemisia capillaris and DE treatment also increased levels of hepatic Cyp2a5 protein. Luciferase reporter assays showed that CAR increases Cyp2a5 gene transcription through a CAR response element in the Cyp2a5 gene promoter. Moreover, DE caused nuclear translocation of CAR in primary mouse hepatocytes and increased Cyp2a5 transcription in the presence of CAR. These results identify a potential CAR-mediated mechanism by which DE regulates Cyp2a5 gene expression and suggests that DE may enhance bilirubin clearance by increasing Cyp2a5 levels. Understanding this process could provide an opportunity for the development of novel therapies for neonatal and other forms of jaundice.

Abstract 244: The Flavonoids Naringenin and Nobiletin Stimulate the AMPKinase Pathway in Primary Mouse Hepatocytes

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Joshua P Samsoondar ◽  
Lazar A Bojic ◽  
Brian G Sutherland ◽  
Gregory R Steinberg ◽  
Jane Y Edwards ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Protein Kinase ◽  
Hepatic Steatosis ◽  
Dependent Manner ◽  
Ampk Activation ◽  
Metabolic Dysregulation ◽  
Malonyl Coa ◽  
Primary Mouse Hepatocytes ◽  
Primary Mouse ◽  
Mouse Hepatocytes

Dyslipidemia associated with insulin resistance and obesity are core features of the metabolic syndrome and type 2 diabetes, which contribute significantly to atherosclerosis. In mouse models of diet-induced metabolic dysregulation, the citrus flavonoids naringenin and nobiletin prevent obesity, hepatic steatosis, apoB100 overproduction, dyslipidemia, insulin resistance and atherosclerosis. To elucidate the mechanism of action in liver we assessed flavonoid-induced activation of AMP-activated protein kinase (AMPK), the major regulator of cellular energy homeostasis, in primary mouse hepatocytes. Stimulated AMPK activity promotes catabolic, ATP-generating processes such as fatty acid (FA) oxidation while inhibiting anabolic processes such as FA synthesis. In primary C57BL/6 (WT) hepatocytes, naringenin and nobiletin increased phosphorylation (P) of AMPK and its downstream target acetyl-CoA carboxylase (ACC) in a time- and dose-dependent manner. This was associated with decreased apoB100 secretion. Phosphorylation of ACC by AMPK inhibits the formation of malonyl-CoA reducing substrate for FA synthesis in the cytosol while relieving inhibition of mitochondrial FA oxidation by malonyl-CoA. Under insulin resistant conditions stimulated by high glucose media, reduced pAMPK and pACC were reversed by flavonoid treatment in WT hepatocytes, whereas these effects were lost in Ampkβ1-/- hepatocytes. Sterol receptor element binding protein-1c, which stimulates lipogenesis, was also phosphorylated (inhibited) by flavonoid-induced AMPK activation. BAPTA, a calcium chelator or STO609, an inhibitor of Ca2+/calmodulin-dependent protein kinase kinase-beta (CaMKKβ), did not block flavonoid-induced pACC, suggesting that CaMKKβ is not required for AMPK activation by flavonoids. In chow-fed Ldlr-/- mice, acute i.p. injection of nobiletin following a fasting-refeeding protocol, depressed the respiratory exchange ratio indicative of a switch to FA oxidation. Freeze-clamped liver samples from these mice sacrificed 90 min. post injection showed marked induction of pAMPK and pACC. These results suggest that naringenin and nobiletin attenuate hepatic steatosis and metabolic dysregulation, in part, through activation of hepatic AMPK.

scholarly journals Docosahexaenoic Acid Ameliorates Fructose-Induced Hepatic Steatosis Involving ER Stress Response in Primary Mouse Hepatocytes

Nutrients ◽  
2016 ◽  
Vol 8 (1) ◽  
pp. 55 ◽  
Author(s):  
Jinying Zheng ◽  
Chuan Peng ◽  
Yanbiao Ai ◽  
Heng Wang ◽  
Xiaoqiu Xiao ◽  
...  
Keyword(s):  
Stress Response ◽  
Docosahexaenoic Acid ◽  
Er Stress ◽  
Hepatic Steatosis ◽  
Er Stress Response ◽  
Primary Mouse Hepatocytes ◽  
Primary Mouse ◽  
Mouse Hepatocytes

miR-27b-3p Down-Regulation Prevents Hypoxia-Induced Cardiomyocyte Apoptosis Through Regulating Yes-Associated Protein 1 (YAP1) Expression

2021 ◽  
Vol 11 (5) ◽  
pp. 948-956
Author(s):  
Lilin Wang ◽  
Bo Feng ◽  
Shu Zhu
Keyword(s):  
Cell Viability ◽  
Target Genes ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
H9c2 Cells ◽  
Luciferase Reporter Gene ◽  
Western Blot Assay ◽  
Heart Protection ◽  
Gene Assay ◽  
Related Proteins

Background: Congenital heart disease (CHD) is one of the most common birth defects. MicroR-NAs (miRNAs) are a group of endogenous, non-coding small RNAs and mediate the target genes expression. An increasing evidence showed that in recent years, miRNAs have given rise to more and more attention in heart protection and development. In our research, the main purpose was to determine the effect of miR-27b-3p in CHD and analyze related mechanisms. Methods: We performed qRT-PCR analysis to examine miR-27b-3p expression in myocardial tissue from 30 patients with CHD and hypoxia-induced H9C2 cells. Then, we performed biological software TargetScan to predict the relationship of miR-27b-3p and YAP1, and dual luciferase reporter gene assay was used to verify the results. H9C2 cells were transfected with inhibitor control, miR-27b-3p inhibitor, miR-27b-3p inhibitor + control-siRNA or miR-27b-3p inhibitor + YAP1-siRNA for 6 hours and then induced by hypoxia for 72 hours. Subsequently, we performed MTT and FCM analysis to detect cell viability and apoptosis. Finally, we used western blot assay to measure the expression of apoptosis-related proteins. Results: Our study indicated that miR-27b-3p expression in myocardial samples of cyanotic CHD patients was significantly higher than that of the acyanotic CHD patients. miR-27b-3p expression was gradually up-regulated with the increase of hypoxia induction time in H9C2 cells. Besides, we confirmed that YAP1 was a target gene of miR-27b-3p. Moreover, our results showed that miR-27b-3p inhibitor improved cell viability, decreased apoptosis, and affected apoptosis-related proteins expression in hypoxia induced H9C2 cells. These changes were reversed by YAP1-siRNA. All data demonstrated that miR-27b-3p/YAP1 might be new potential bio-marker and therapeutic target for CHD treatment.

PLAGL2 Cooperates in Leukemia Development by Direcly Inducing c-Mpl Expression.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 744-744
Author(s):  
Sean F. Landrette ◽  
Lucio H. Castilla
Keyword(s):  
Target Genes ◽  
Receptor Expression ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Donor Cells ◽  
Human Acute Myeloid Leukemia ◽  
Reporter Assays ◽  
Leukemia Development ◽  
Upstream Regulators

Abstract Inversion of chromosome 16 is found in 12% of human acute myeloid leukemia (AML). This inversion creates the CBFΒ-SMMHC fusion protein, which hinders myeloid differentiation and triggers AML in collaboration with additional mutations. We have previously shown that the zinc finger transcription factor PLAGL2 efficiently cooperates with CbfΒ-SMMHC in murine AML, and that PLAGL2 transcript levels are significantly increased in inversion 16 human AML. The mechanism of PLAGL2 action in AML, however, is not understood. In order to identify candidate PLAGL2 target genes in AML, we performed gene expression profiling of primary hematopoietic progenitors expressing CbfΒ-SMMHC and PLAGL2, as well as in AML samples induced by CbfΒ-SMMHC and PLAGL2. The expression of c-Mpl transcript was increased 8 to 32 fold in progenitors and AML samples when compared to controls not expressing PLAGL2. This increase was confirmed by quantitative PCR analysis and correlated with increased c-Mpl receptor in membrane by flow cytometry. Analysis of the c-Mpl proximal promoter identified three conserved Plag binding sites. PLAGL2 binding to these sites was confirmed by chromatin immunoprecipitation and luciferase reporter assays. Furthermore, PLAGL2/CbfΒ-SMMHC AML cells show a significant increase in sensitivity to the c-Mpl-ligand thrombopoietin when compared to non-PLAGL2 CbfΒ-SMMHC-AML samples as measured by Jak2 phosphorylation. To test whether PLAGL2 induces AML by increasing c-Mpl levels, AML development was analyzed in bone-marrow transplantation assays using CbfΒ-SMMHC-expressing donor cells transduced with c-Mpl expressing retrovirus. All transplanted mice (8/8) succumbed to leukemia with a median latency of 8 weeks, while control groups remained unaffected for 16 weeks. The pathology of Mpl/CbfΒ-SMMHC AML was immuno-phenotypically and morphologically identical to that of PLAGL2/CbfΒ-SMMHC AML. This study demonstrates that PLAGL2 directly induces c-Mpl receptor expression, and that such increase cooperates with CbfΒ-SMMHC in AML development. These results suggest that AML can result from the alteration of upstream regulators of Mpl/Jak2 signaling.

scholarly journals Exosomal miR-21 regulates the TETs/PTENp1/PTEN pathway to promote hepatocellular carcinoma growth

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Liang-qi Cao ◽  
Xue-wei Yang ◽  
Yu-bin Chen ◽  
Da-wei Zhang ◽  
Xiao-Feng Jiang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Target Genes ◽  
Underlying Mechanism ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Reporter Assays ◽  
Important Means ◽  
And Migration ◽  
Insight Into ◽  
Hcc Cells

Abstract Background As an important means of communication, exosomes play an important role in the development of hepatocellular carcinoma (HCC). Methods Bioinformatics analysis, dual-luciferase reporter assays, methylation-specific quantitative PCR, and ChIP-PCR analysis were used to gain insight into the underlying mechanism of miR-21 in HCC. Results The detection of miRNAs in exosomes of HCC showed that miR-21 expression in exosomes was positively correlated with the expression level of miR-21 in cells and negatively correlated with the expression of its target genes PTEN, PTENp1 and TETs. HCC cell-derived exosomes could increase miR-21 and p-Akt expression in HCC cells and downregulate the expression of PTEN, PTENp1 and TETs. MiR-21 inhibitors or PTENp1 overexpression vectors could weaken the effect of the abovementioned exosomes and simultaneously weaken their role in promoting cell proliferation and migration and inhibiting apoptosis. Further studies showed that miR-21 not only directly regulated the expression of PTEN, PTENp1 and TETs but also increased the methylation level of the PTENp1 promoter by regulating the expression of TETs, thereby inhibiting the expression of PTENp1 and further downregulating the expression of PTEN. Conclusions Exosomal miR-21 can regulate the expression of the tumor suppressor genes PTEN and PTENp1 in various ways and affect the growth of HCC cells.

scholarly journals Role of Toll-Like Receptors in Changes in Gene Expression and NF-κB Activation in Mouse Hepatocytes Stimulated with Lipopolysaccharide

2002 ◽  
Vol 70 (7) ◽  
pp. 3433-3442 ◽  
Author(s):  
Shubing Liu ◽  
David J. Gallo ◽  
Angela M. Green ◽  
Debra L. Williams ◽  
Xiaoyan Gong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Map Kinase ◽  
Global Gene Expression ◽  
Pcr Analysis ◽  
Toll Like Receptors ◽  
Kinase Activation ◽  
Extracellular Signal ◽  
Primary Mouse ◽  
Mitogen Activated Protein ◽  
Mouse Hepatocytes

ABSTRACT The liver is an important site of host-microbe interaction. Although hepatocytes have been reported to be responsive to lipopolysaccharide (LPS), the global gene expression changes by LPS and mechanism(s) by which LPS stimulates cultured hepatocytes remain uncertain. Cultures of primary mouse hepatocytes were incubated with LPS to assess its effects on the global gene expression, hepatic transcription factors, and mitogen-activated protein (MAP) kinase activation. DNA microarray analysis indicated that LPS modulates the selective expression of more than 80 genes and expressed sequence tags. We have shown previously that hepatocytes express CD14, which is required both for uptake and responsiveness to LPS. In other cells, responsiveness to microbial products requires expression of Toll-like receptors (TLR) and their associated accessory molecules. Hepatocytes expressed TLR1 through TLR9 as well as MyD88 and MD-2 transcripts, as shown by reverse transcriptase PCR analysis, indicating that hepatocytes express all known microbe recognition molecules. The MAP kinase extracellular signal-regulated kinase 1/2 was phosphorylated in response to LPS in mouse hepatocytes, and the levels of phosphorylation were lower in hepatocytes from TLR4-null mice. NF-κB activation was reduced in TLR4-mutant or -null hepatocytes compared to control hepatocytes, and this defect was partially restored by adenoviral transduction of mouse TLR4. Thus, hepatocytes respond to nanogram concentrations of LPS through a TLR4 response pathway.

Sign in / Sign up

Export Citation Format

Share Document