scholarly journals Translational repression of SLC26A3 by miR-494 in intestinal epithelial cells

2014 ◽  
Vol 306 (2) ◽  
pp. G123-G131 ◽  
Author(s):  
Arivarasu N. Anbazhagan ◽  
Shubha Priyamvada ◽  
Anoop Kumar ◽  
Daniel B. Maher ◽  
Alip Borthakur ◽  
...  
Keyword(s):  
Protein Expression ◽  
Therapeutic Strategy ◽  
Luciferase Activity ◽  
Negative Control ◽  
Intestinal Epithelial ◽  
Diarrheal Diseases ◽  
Regulate Protein ◽  
Putative Binding Site ◽  
Mammalian Intestine ◽  
Relative Luciferase Activity

SLC26A3 [downregulated in adenoma (DRA)] is a Cl−/HCO3− exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Altered DRA expression levels are associated with infectious and inflammatory diarrheal diseases. Therefore, it is critical to understand the regulation of DRA expression. MicroRNAs (miRNAs) are endogenous, small RNAs that regulate protein expression via blocking the translation and/or promoting mRNA degradation. To investigate potential modulation of DRA expression by miRNA, five different in silico algorithms were used to predict the miRNAs that target DRA. Of these miRNAs, miR-494 was shown to have a highly conserved putative binding site in the DRA 3′-untranslated region (3′-UTR) compared with other DRA-targeting miRNAs in vertebrates. Transfection with pmirGLO dual luciferase vector containing DRA 3′-UTR (pmirGLO-3′-UTR DRA) resulted in a significant decrease in relative luciferase activity compared with empty vector. Cotransfection of the DRA 3′-UTR luciferase vector with a miR-494 mimic further decreased luciferase activity compared with cells transfected with negative control. The transfection of a miR-494 mimic into Caco-2 and T-84 cells significantly increased the expression of miR-494 and concomitantly decreased the DRA protein expression. Mutation of the seed sequences for miR-494 in 3′-UTR of DRA abrogated the effect of miR-494 on 3′-UTR. These data demonstrate a novel regulatory mechanism of DRA expression via miR-494 and indicate that targeting this microRNA may serve to be a potential therapeutic strategy for diarrheal diseases.

scholarly journals Role of MiR-155 Signal Pathway in Regulating Podocyte Injury Induced by TGF-β1

2017 ◽  
Vol 42 (4) ◽  
pp. 1469-1480 ◽  
Author(s):  
Xu Lin ◽  
Xintng Zhen ◽  
Haiting Huang ◽  
Haohao Wu ◽  
Yanwu You ◽  
...  
Keyword(s):  
Protein Expression ◽  
Antisense Oligonucleotides ◽  
Negative Control ◽  
Signal Pathway ◽  
Caspase 9 ◽  
Podocyte Injury ◽  
Apoptosis Rate ◽  
Mrna And Protein Expression ◽  
Tgf Β1

Background/Aims: Transforming growth factor beta 1 (TGF-β1) plays a critical role in the pathogenesis of glomerulosclerosis. The purpose of this study was to examine the effects of inhibition of miR-155 on podocyte injury induced by TGF-β1 and to determine further molecular mediators involved in the effects of miR-155. Methods: Conditionally immortalized podocytes were cultured in vitro and they were divided into four groups: control; TGF-β1 treatment; TGF-β1 with miR-155 knockdown [using antisense oligonucleotides against miR-155 (ASO-miR-155)] and TGF-β1 with negative control antisense oligonucleotides (ASO-NC). Real time RT-PCR and Western blot analysis were employed to determine the mRNA and protein expression of nephrin, desmin and caspase-9, respectively. Flow cytometry was used to examine the apoptotic rate of podocytes and DAPI fluorescent staining was used to determine apoptotic morphology. In addition, we examined the levels of miR-155, TGF-β1, nephrin, desmin and caspase-9 in glomerular tissues of nephropathy induced by intravenous injections of adriamycin in rats. Results: mRNA and protein expression of desmin and caspase-9 was increased in cultured TGF-β1-treated podocytes, whereas nephrin was decreased as compared with the control group. Importantly, miR-155 knockdown significantly attenuated upregulation of desmin and caspase-9, and alleviated impairment of nephrin induced by TGF-β1. Moreover, the number of apoptotic podocytes was increased after exposure to TGF-β1 and this was alleviated after miR-155 knockdown. Knocking down miR-155 also decreased an apoptosis rate of TGF-β1-treated podocytes. Note that negative control antisense oligonucleotides failed to alter an increase of the apoptosis rate in TGF-β1-treated podocytes. Consistent with in vitro results, expression of miR-155, TGF-β1, desmin and caspase-9 was increased and nephrin was decreased in glomerular tissues with nephropathy in vivo experiments. Conclusions: TGF-β1 impairs the protein expression of nephrin and amplifies the protein expression of desmin and caspase -9 via miR-155 signal pathway. Inhibition of miR-155 alleviates these changes in podocytes-treated with TGF-β1 and attenuated apoptosis of podocytes. Our data suggest that miR-155 plays a role in mediating TGF-β1-induced podocyte injury via nephrin, desmin and caspase-9. Results of the current study also indicate that blocking miR-155 signal has a protective effect on podocyte injury. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of podocyte injury observed in glomerulosclerosis.

scholarly journals Adhesive properties of an outer structure of Clostridium perfringens type A isolated from piglets with with catarrhal enteritis

1999 ◽  
Vol 30 (3) ◽  
pp. 242-248 ◽  
Author(s):  
Elizabeth Pelosi Teixeira ◽  
Marlene Braide Serafim ◽  
Maria Alice Cruz Höfling ◽  
Aureo T. Yamada ◽  
Antonio Fernando Pestana de Castro
Keyword(s):  
Clostridium Perfringens ◽  
Type A ◽  
Negative Control ◽  
Bacterial Cells ◽  
Intestinal Epithelial ◽  
Adhesive Properties ◽  
Ileal Loop ◽  
Fibrillar Material ◽  
Gram Staining ◽  
Transmission Electron

One strain (S32) of Clostridium perfringens type A was isolated from a case of catarrhal enteritis of piglets. This strain was able to adhere to HeLa cells showing an adherence index (AI) of 25.15 ± 1.26 (mean ± 1 standard error of the mean). Treatment of the bacterial cells with trypsin (0.25mg/ml) decreased in 70%-80% the AI and metaperiodate (10mg/ml) abolished completely the adherence, suggesting that the structure responsible for this phenomenon was probably a glycoprotein. Heating of bacterial suspensions (100ºC/5 min) before carrying out the adhesion test decreased the AI rendering it equal to the negative controls. Rabbit homologous S32 antiserum inhibited the adherence up to dilutions of 1: 640, at least. The piglet ileal loop assay, carried out with strains S32 and Jab-1 (negative control) demonstrated that the strain S32 was able to adhere to the intestinal epithelial cells when examined after Gram staining. Transmission electron microcopy (TEM) demonstrated that S32 strain displayed a loose fibrillar material not seen with Jab-1. Stabilization of the bacterial cells with homologous antiserum of strain S32, followed by staining with rhuteniun red, revealed loose long fibrillar material on the outer surface of the cells, that sometimes could be seen spreading out from the cells and linking bacterial cells. The question whether this structure might be an adhesin for this strain of Cl. perfringes type A, perhaps playing a role in the pathogenesis of the catarrhal enteritis of piglets, is dependent on further studies.

scholarly journals Thyrotrophin receptor protein expression in normal and adenomatous human pituitary

2000 ◽  
Vol 167 (1) ◽  
pp. 7-13 ◽  
Author(s):  
M Theodoropoulou ◽  
T Arzberger ◽  
Y Gruebler ◽  
Z Korali ◽  
P Mortini ◽  
...  
Keyword(s):  
Protein Expression ◽  
Pituitary Adenomas ◽  
Anterior Pituitary ◽  
Preliminary Evidence ◽  
Positive Control ◽  
Negative Control ◽  
Receptor Protein ◽  
Human Pituitary ◽  
Thyrotrophin Releasing Hormone ◽  
Regulatory Pathways

Thyrotrophin (TSH) synthesis and secretion is under the positive control of thyrotrophin releasing hormone and under the negative control of the thyroid hormones. However, it is hypothesised that TSH has a direct effect on the regulation of its own synthesis through an intrapituitary loop mediated by pituitary TSH receptors (TSH-R). The aim of this investigation was to study the expression of TSH-R in normal human pituitary at mRNA and protein levels, and to compare the pattern of protein expression between different pituitary adenomas. Using RT-PCR we were able to detect TSH-R mRNA in the normal pituitary, and immunohistochemical studies showed TSH-R protein expression in distinct areas of the anterior pituitary. Double immunostaining with antibodies against each of the intrapituitary hormones and S100 revealed that TSH-R protein is present in thyrotrophs and folliculostellate cells. Examination of 58 pituitary adenomas, including two clinically active and two clinically inactive thyrotroph adenomas, revealed TSH-R immunopositivity in only the two clinically inactive thyrotroph adenomas. This study shows, for the first time, the presence of TSH-R protein in the normal anterior pituitary and in a subset of thyrotroph adenomas. The expression of TSH-R in the thyrotroph and folliculostellate cell subpopulations provides preliminary evidence of a role for TSH in autocrine and paracrine regulatory pathways within the anterior pituitary gland.

Abstract 239: MicroRNAs hsa-miR-584 and hsa-miR-31 Regulate Expression of Human Angiotensnogen Gene

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Brahmaraju Mopidevi ◽  
Shreekrishna Maharjan ◽  
Sudhir Jain ◽  
Varunkumar G. Pandey ◽  
Ashok Kumar
Keyword(s):  
Luciferase Activity ◽  
Negative Control ◽  
Hek293 Cells ◽  
Multiple Cloning Site ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Hep3b Cells ◽  
Agt Gene ◽  
Dose Dependent ◽  
Mirna Expression Vector

Background: Hypertension is a risk factor for stroke, myocardial infarction, and congestive heart failure. A single nucleotide polymorphism (SNP) (rs7079) in 3’UTR of human angiotensinogen (hAGT) gene is associated with elevated blood pressure. We have used TargetScan V6.0, miRanda miRNA prediction algorithms and found that two microRNAs hsa-miR-584 and hsa-miR-31 may bind differentially to rs7079 and alter the expression of hAGT gene. METHODS: The effect of hsa-miR-584 and hsa-miR-31 miRNAs on endogenous AGT expression levels in Hep3B cells were studied by transfection of individual miRNA mimics followed by quantitative real time PCR (Q-RT-PCR) of the hAGT gene. In addition, the 600 bp 3’UTR of hAGT gene containing either rs7079C or rs7079A was PCR amplified and cloned in to the multiple cloning site of pMIR-REPORT™ miRNA Expression Vector containing luciferase gene. These reporter constructs were then co-transfected with either miRNA mimics or inhibitors to study the effect of miRNAs on luciferase activity in Hep3B cells since these cells express hAGT gene. These experiments were also performed in HEK293 cells which do not express the hAGT gene. Results: Q-RT-PCR showed that hsa-miR-584 and hsa-miR-31 mimics at 50nM concentration reduced endogenous hAGT mRNA levels by 38% and 30% respectively compared to the mock (without any microRNA) or negative control (which does not have any binding site for eukaryotic 3’UTRs) in Hep3B cells. Furthermore hsa-miR-584 and hsa-miR-31 showed 40% and 25% reduced luciferase activity of the construct containing rs7079 C allele. On the other hand these miRNAs did not affect the luciferase activity in the presence of rs7079 A. When dose dependent anti miRNA inhibitors were transfected along with miRNA mimics, these mimics abolished the microRNA induced down-regulation of luciferase activity. Conclusion: hsa-miR-584 and hsa-miR-31 miRNAs bind strongly to the hAGT 3’UTR containing rs7079 C allele as compared to rs7079 A allele and may down regulate the expression of AGT gene containing rs7079 C allele. This may be one of the possible mechanism involved in association of rs7079 A allele with human hypertension.

scholarly journals Interferon-γ inhibits sirtuin 6 gene expression in intestinal epithelial cells through a microRNA-92b-dependent mechanism

2020 ◽  
Vol 318 (4) ◽  
pp. C732-C739
Author(s):  
Fangyi Liu ◽  
Xiao Wang ◽  
Hua Geng ◽  
Heng-Fu Bu ◽  
Peng Wang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Protein Expression ◽  
Intestinal Epithelial Cells ◽  
Specific Inhibitor ◽  
In Silico Analysis ◽  
Interferon Γ ◽  
Luciferase Assay ◽  
Epithelial Cell Line ◽  
Dependent Manner ◽  
Intestinal Epithelial

Sirtuin 6 (Sirt6) is predominantly expressed in epithelial cells in intestinal crypts. It plays an important role in protecting intestinal epithelial cells against inflammatory injury. Previously, we found that colitis is associated with the downregulation of Sirt6 protein in the intestines. Here, we report that murine interferon-γ (Ifnγ) inhibits Sirt6 protein but not mRNA expression in young adult mouse colonocytes (YAMC, a mouse colonic epithelial cell line) in a dose- and time-dependent manner. Using microRNA array analysis, we showed that Ifnγ induces expression of miR-92b in YAMC cells. With in silico analysis, we found that the Sirt6 3′-untranslated region (UTR) contains a putative binding site for miR-92b. Luciferase assay showed that Ifnγ inhibited Sirt6 3′-UTR activity and this effect was mimicked by miR-92b via directly targeting the miR-92b seed site in the 3′-UTR of Sirt6 mRNA. Furthermore, Western blot demonstrated that miR-92b downregulated Sirt6 protein expression in YAMC cells. Blocking miR-92b with a specific inhibitor attenuated the inhibitory effect of Ifnγ on Sirt6 protein expression in the cells. Collectively, our data suggest that Ifnγ inhibits Sirt6 protein expression in intestinal epithelial cells via a miR-92b-mediated mechanism. miR-92b may be a novel therapeutic target for rescuing Sirt6 protein levels in intestinal epithelial cells, thereby protecting against intestinal mucosal injury caused by inflammation.

Extracellular matrix proteins differentially regulate airway smooth muscle phenotype and function

2007 ◽  
Vol 292 (6) ◽  
pp. L1405-L1413 ◽  
Author(s):  
Bart G. J. Dekkers ◽  
Dedmer Schaafsma ◽  
S. Adriaan Nelemans ◽  
Johan Zaagsma ◽  
Herman Meurs
Keyword(s):  
Smooth Muscle ◽  
Protein Expression ◽  
Airway Smooth Muscle ◽  
Airway Remodeling ◽  
Negative Control ◽  
Collagen I ◽  
Contractile Protein ◽  
Chronic Obstructive ◽  
Ecm Proteins ◽  
And Function

Changes in the ECM and increased airway smooth muscle (ASM) mass are major contributors to airway remodeling in asthma and chronic obstructive pulmonary disease. It has recently been demonstrated that ECM proteins may differentially affect proliferation and expression of phenotypic markers of cultured ASM cells. In the present study, we investigated the functional relevance of ECM proteins in the modulation of ASM contractility using bovine tracheal smooth muscle (BTSM) preparations. The results demonstrate that culturing of BSTM strips for 4 days in the presence of fibronectin or collagen I depressed maximal contraction (Emax) both for methacholine and KCl, which was associated with decreased contractile protein expression. By contrast, both fibronectin and collagen I increased proliferation of cultured BTSM cells. Similar effects were observed for PDGF. Moreover, PDGF augmented fibronectin- and collagen I-induced proliferation in an additive fashion, without an additional effect on contractility or contractile protein expression. The fibronectin-induced depression of contractility was blocked by the integrin antagonist Arg-Gly-Asp-Ser (RGDS) but not by its negative control Gly-Arg-Ala-Asp-Ser-Pro (GRADSP). Laminin, by itself, did not affect contractility or proliferation but reduced the effects of PDGF on these parameters. Strong relationships were found between the ECM-induced changes in Emax in BTSM strips and their proliferative responses in BSTM cells and for Emax and contractile protein expression. Our results indicate that ECM proteins differentially regulate both phenotype and function of intact ASM.

LPA protects intestinal epithelial cells from apoptosis by inhibiting the mitochondrial pathway

2003 ◽  
Vol 284 (5) ◽  
pp. G821-G829 ◽  
Author(s):  
Wenlin Deng ◽  
De-An Wang ◽  
Elvira Gosmanova ◽  
Leonard R. Johnson ◽  
Gabor Tigyi
Keyword(s):  
Epithelial Cells ◽  
Cytochrome C ◽  
Protein Expression ◽  
Caspase 3 ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Caspase 9 ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
Antiapoptotic Activity

We previously showed ( Gastroenterology 123: 206–216, 2002) that lysophosphatidic acid (LPA) protects and rescues rat intestinal epithelial cells (IEC-6) from apoptosis. Here, we provide evidence for the LPA-elicited inhibition of the mitochondrial apoptotic pathway leading to attenuation of caspase-3 activation. Pretreatment of IEC-6 cells with LPA inhibited campothecin-induced caspase-9 and caspase-3 activation and DNA fragmentation. A caspase-9 inhibitor peptide mimicked the LPA-elicited antiapoptotic activity. LPA elicited ERK1/ERK2 and PKB/Akt phosphorylation. The LPA-elicited antiapoptotic activity and inhibition of caspase-9 activity were abrogated by pertussis toxin, PD 98059, wortmannin, and LY 294002. LPA reduced cytochrome c release from mitochondria and prevented activation of caspase-9. LPA prevented translocation of Bax from cytosol to mitochondria and increased the expression of the antiapoptotic Bcl-2 mRNA and protein. LPA had no effect on Bcl-xl, Bad, and Bak mRNA or protein expression. These data indicate that LPA protects IEC-6 cells from camptothecin-induced apoptosis through Gi-coupled inhibition of caspase-3 activation mediated by the attenuation of caspase-9 activation due to diminished cytochrome c release, involving upregulation of Bcl-2 protein expression and prevention of Bax translocation.

scholarly journals Different Sources of Copper Effect on Intestinal Epithelial Cell: Toxicity, Oxidative Stress, and Metabolism

Metabolites ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 11
Author(s):  
Runxian Li ◽  
Yang Wen ◽  
Gang Lin ◽  
Chengzhen Meng ◽  
Pingli He ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Epithelial Cell ◽  
Protein Expression ◽  
Intestinal Epithelial Cell ◽  
Cell Damage ◽  
Intestinal Epithelial ◽  
Protein Expression Level ◽  
Treatment Groups ◽  
All Treatment

Copper (Cu) is widely used in the swine industry to improve the growth performance of pigs. However, high doses of copper will induce cell damage and toxicity. The aim of this study was to evaluate toxicity, bioavailability, and effects on metabolic processes of varying copper sources using porcine intestinal epithelial cells (IPEC-J2) as a model. The IPEC-J2 were treated with two doses (30 and 120 μM) of CuSO4, Cu Glycine (Cu-Gly), and Cu proteinate (Cu-Pro) for 10 h, respectively. Cell damage and cellular copper metabolism were measured by the changes in cell viability, copper uptake, oxidative stress biomarkers, and gene/protein expression levels. The results showed that cell viability and ratio of reduced and oxidized glutathione (GSH/GSSG) decreased significantly in all treatment groups; intracellular copper content increased significantly in all treatment groups; total superoxide dismutase (SOD) activity increased significantly in the 120 μM exposed groups; SOD1 protein expression levels were significantly upregulated in 30 μM Cu-Pro, 120 μM Cu-Gly, and 120 μM Cu-Pro treatment groups; intracellular reactive oxygen species (ROS) generation and malondialdehyde (MDA) content increased significantly in 30 μM treatment groups and 120 μM CuSO4 treatment group. CTR1 and ATP7A gene expression were significantly downregulated in the 120 μM exposed groups. While upregulation of ATOX1 expression was observed in the presence of 120 μM Cu-Gly and Cu-Pro. ASCT2 gene expression was significantly upregulated after 120 μM Cu-Glycine and CuSO4 exposure, and PepT1 gene expression was significantly upregulated after Cu-Pro exposure. In addition, CTR1 protein expression level decreased after 120 μM CuSO4 and Cu-Gly exposure. PepT1 protein expression level was only upregulated after 120 μM Cu-Pro exposure. These findings indicated that extra copper supplementation can induce intestinal epithelial cell injury, and different forms of copper may have differing effects on cell metabolism.

Mechanism of TNF-α modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression

2005 ◽  
Vol 288 (3) ◽  
pp. G422-G430 ◽  
Author(s):  
Thomas Y. Ma ◽  
Michel A. Boivin ◽  
Dongmei Ye ◽  
Ali Pedram ◽  
Hamid M. Said
Keyword(s):  
Protein Expression ◽  
Tight Junction ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial ◽  
Mrna Transcription ◽  
Tnf Α ◽  
Epithelial Tight Junction

TNF-α plays a central role in the intestinal inflammation of various inflammatory disorders including Crohn's disease (CD). TNF-α-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed as one of the proinflammatory mechanisms contributing to the intestinal inflammation. The intracellular mechanisms involved in the TNF-α-induced increase in intestinal TJ permeability remain unclear. The purpose of this study was to investigate the possibility that the TNF-α-induced increase in intestinal epithelial TJ permeability was regulated by myosin light-chain kinase (MLCK) protein expression, using an in vitro intestinal epithelial model system consisting of the filter-grown Caco-2 intestinal epithelial monolayers. TNF-α (10 ng/ml) produced a time-dependent increase in Caco-2 MLCK expression. The TNF-α increase in MLCK protein expression paralleled the increase in Caco-2 TJ permeability, and the inhibition of the TNF-α-induced MLCK expression (by cycloheximide) prevented the increase in Caco-2 TJ permeability, suggesting that MLCK expression may be required for the increase in Caco-2 TJ permeability. The TNF-α increase in MLCK protein expression was preceded by an increase in MLCK mRNA expression but not an alteration in MLCK protein degradation. Actinomycin-D prevented the TNF-α increase in MLCK mRNA expression and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability, suggesting that the increase in MLCK mRNA transcription led to the increase in MLCK expression. The TNF-α increase in MLCK protein expression was also associated with an increase in Caco-2 MLCK activity. The cycloheximide inhibition of MLCK protein expression prevented the TNF-α increase in MLCK activity and Caco-2 TJ permeability. Moreover, inhibitors of MLCK, Mg2+-myosin ATPase, and metabolic energy prevented the TNF-α increase in Caco-2 TJ permeability, suggesting that the increase in MLCK activity was required for the TNF-α-induced opening of the Caco-2 TJ barrier. In conclusion, our results indicate for the first time that 1) the TNF-α increase in Caco-2 TJ permeability was mediated by an increase in MLCK protein expression, 2) the increase in MLCK protein expression was regulated by an increase in MLCK mRNA transcription, and 3) the increase in Caco-2 TJ permeability required MLCK protein expression-dependent increase in MLCK activity.

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