scholarly journals Allele-Specific MicroRNA-Mediated Regulation of a Glycolysis Gatekeeper PDK1 in Cancer Metabolism

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3582
Author(s):  
Sugarniya Subramaniam ◽  
Varinder Jeet ◽  
Jennifer H. Gunter ◽  
Judith A. Clements ◽  
Jyotsna Batra
Keyword(s):  
Reporter Gene ◽  
Cancer Metabolism ◽  
In Silico Analysis ◽  
Pyruvate Dehydrogenase Kinase ◽  
Dependent Manner ◽  
Protein Levels ◽  
Gene Assay ◽  
Allele Specific ◽  
Pyruvate Dehydrogenase Kinase 1 ◽  
In Cells

Background: Emerging evidence has revealed that genetic variations in microRNA (miRNA) binding sites called miRSNPs can alter miRNA binding in an allele-specific manner and impart prostate cancer (PCa) risk. Two miRSNPs, rs1530865 (G > C) and rs2357637 (C > A), in the 3′ untranslated region of pyruvate dehydrogenase kinase 1 (PDK1) have been previously reported to be associated with PCa risk. However, these results have not been functionally validated. Methods: In silico analysis was used to predict miRNA–PDK1 interactions and was tested using PDK1 knockdown, miRNA overexpression and reporter gene assay. Results: PDK1 expression was found to be upregulated in PCa metastasis. Further, our results show that PDK1 suppression reduced the migration, invasion, and glycolysis of PCa cells. Computational predictions showed that miR-3916, miR-3125 and miR-3928 had a higher binding affinity for the C allele than the G allele for the rs1530865 miRSNP which was validated by reporter gene assays. Similarly, miR-2116 and miR-889 had a higher affinity for the A than C allele of the rs2357637 miRSNP. Overexpression of miR-3916 and miR-3125 decreased PDK1 protein levels in cells expressing the rs1530865 SNP C allele, and miR-2116 reduced in cells with the rs2357637 SNP A allele. Conclusions: The present study is the first to report the regulation of the PDK1 gene by miRNAs in an allele-dependent manner and highlights the role of PDK1 in metabolic adaption associated with PCa progression.

scholarly journals Suppression of Pyruvate Dehydrogenase Kinase by Dichloroacetate in Cancer and Skeletal Muscle Cells Is Isoform Specific and Partially Independent of HIF-1α

2021 ◽  
Vol 22 (16) ◽  
pp. 8610
Author(s):  
Nives Škorja Milić ◽  
Klemen Dolinar ◽  
Katarina Miš ◽  
Urška Matkovič ◽  
Maruša Bizjak ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cancer Cells ◽  
Pyruvate Dehydrogenase ◽  
Hypoxia Inducible Factor ◽  
Muscle Cells ◽  
Pyruvate Dehydrogenase Kinase ◽  
Skeletal Muscle Cells ◽  
Dependent Manner ◽  
Protein Levels ◽  
L6 Myotubes

Inhibition of pyruvate dehydrogenase kinase (PDK) emerged as a potential strategy for treatment of cancer and metabolic disorders. Dichloroacetate (DCA), a prototypical PDK inhibitor, reduces the abundance of some PDK isoenzymes. However, the underlying mechanisms are not fully characterized and may differ across cell types. We determined that DCA reduced the abundance of PDK1 in breast (MDA-MB-231) and prostate (PC-3) cancer cells, while it suppressed both PDK1 and PDK2 in skeletal muscle cells (L6 myotubes). The DCA-induced PDK1 suppression was partially dependent on hypoxia-inducible factor-1α (HIF-1α), a transcriptional regulator of PDK1, in cancer cells but not in L6 myotubes. However, the DCA-induced alterations in the mRNA and the protein levels of PDK1 and/or PDK2 did not always occur in parallel, implicating a role for post-transcriptional mechanisms. DCA did not inhibit the mTOR signaling, while inhibitors of the proteasome or gene silencing of mitochondrial proteases CLPP and AFG3L2 did not prevent the DCA-induced reduction of the PDK1 protein levels. Collectively, our results suggest that DCA reduces the abundance of PDK in an isoform-dependent manner via transcriptional and post-transcriptional mechanisms. Differential response of PDK isoenzymes to DCA might be important for its pharmacological effects in different types of cells.

scholarly journals PHD2 Is a Regulator for Glycolytic Reprogramming in Macrophages

2016 ◽  
Vol 37 (1) ◽  
Author(s):  
Annemarie Guentsch ◽  
Angelika Beneke ◽  
Lija Swain ◽  
Katja Farhat ◽  
Shunmugam Nagarajan ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Critical Role ◽  
Hypoxia Inducible Factor ◽  
Pyruvate Dehydrogenase Kinase ◽  
Metabolic Phenotype ◽  
Protein Levels ◽  
Dehydrogenase Enzyme ◽  
Pyruvate Dehydrogenase Kinase 1 ◽  
And Function

ABSTRACT The prolyl-4-hydroxylase domain (PHD) enzymes are regarded as the molecular oxygen sensors. There is an interplay between oxygen availability and cellular metabolism, which in turn has significant effects on the functionality of innate immune cells, such as macrophages. However, if and how PHD enzymes affect macrophage metabolism are enigmatic. We hypothesized that macrophage metabolism and function can be controlled via manipulation of PHD2. We characterized the metabolic phenotypes of PHD2-deficient RAW cells and primary PHD2 knockout bone marrow-derived macrophages (BMDM). Both showed typical features of anaerobic glycolysis, which were paralleled by increased pyruvate dehydrogenase kinase 1 (PDK1) protein levels and a decreased pyruvate dehydrogenase enzyme activity. Metabolic alterations were associated with an impaired cellular functionality. Inhibition of PDK1 or knockout of hypoxia-inducible factor 1α (HIF-1α) reversed the metabolic phenotype and impaired the functionality of the PHD2-deficient RAW cells and BMDM. Taking these results together, we identified a critical role of PHD2 for a reversible glycolytic reprogramming in macrophages with a direct impact on their function. We suggest that PHD2 serves as an adjustable switch to control macrophage behavior.

scholarly journals Tyrosine Phosphorylation of Mitochondrial Pyruvate Dehydrogenase Kinase 1 Is Important for Cancer Metabolism

2011 ◽  
Vol 44 (6) ◽  
pp. 864-877 ◽  
Author(s):  
Taro Hitosugi ◽  
Jun Fan ◽  
Tae-Wook Chung ◽  
Katherine Lythgoe ◽  
Xu Wang ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Pyruvate Dehydrogenase ◽  
Cancer Metabolism ◽  
Pyruvate Dehydrogenase Kinase ◽  
Pyruvate Dehydrogenase Kinase 1

A Role for Splicing Factors and the Transcriptional Machinery in the Pathophysiology of Fanconi Anemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3443-3443
Author(s):  
Allison M Green ◽  
James B Wilson ◽  
Nigel J Jones ◽  
Gary M. Kupfer
Keyword(s):  
Dna Damage ◽  
Fanconi Anemia ◽  
Genetic Disorder ◽  
Direct Interaction ◽  
Congenital Defects ◽  
Splicing Factors ◽  
Dependent Manner ◽  
Protein Levels ◽  
Transcriptional Machinery ◽  
In Cells

Abstract Abstract 3443 Introduction: Fanconi anemia(FA) is an autosomal and X-linked recessive genetic disorder characterized by congenital defects, aplastic anemia, and a predisposition to cancer. At the cellular level, patients with FA display hypersensitivity to DNA crosslinking agents and increased levels of chromosomal instability. Because of these cellular phenotypes, the FA pathway has been thought to function in DNA damage repair response. Our data suggest that one of the ways in which the FA pathway maintains genomic stability within cells is by preventing aberrant transcription following DNA damage. This regulation may be accomplished through a connection to splicing factors or through direct interaction with the transcriptional machinery itself (RNAPII). Methods: siRNA transfections were used to reduce protein levels of splicing factors. Survival assays were performed to determine cell sensitivity to mitomycin C (MMC). Chromatin extractions were performed to determine chromatin loading of proteins. Immunoprecipitations were used to determine protein-protein interactions. Results: Previous studies in our lab have shown that siRNA mediated knock down of the splicing factors ASF/SF2 and SC35 but not SRp55 result in decreased levels of FANCD2 ubiquitylation following MMC treatment and hypersensitivity to MMC. We have since shown that depletion of ASF/SF2 and SC35 but not SRp55 also prevents proper FANCD2 chromatin loading following MMC treatment. As depletion of the splicing factor ASF/SF2 has also been demonstrated to increase cellular genomic instability through the formation of increased levels of R-loop structures, we next wanted to determine what effects increased levels of RNaseH would have on activation of the FA pathway in cells with decreased ASF/SF2 expression. Interestingly, RNaseH overexpression was able to partially rescue the decreased levels of FANCD2 ubiquitylation following MMC treatment and hypersensitivity to MMC seen in cells with decreased protein levels of ASF/SF2. As splicing has been shown to occur co-transcriptionally, we next went on to determine whether transcription occurs normally in cells lacking an intact FA pathway. We discovered that proper degradation of the hyperphosphorylated, transcription competent form of RNAPII in response to DNA damage is dependent upon an intact FA pathway as cells mutant in FANCA or FANCD2 show delayed RNAPII degradation following MMC treatment. Accordingly, we also saw a decrease in the levels of RNAPII interacting with FANCD2 in chromatin after similar, short term MMC treatments. This was accompanied by FANCD2 interaction with RNAPII and BARD1 in a FANCD2 ubiquitylation-dependent manner. Conclusions: These results suggest that the FA pathway may play a part in regulating transcription via a connection to splicing factors and through direct interaction with the transcriptional machinery itself as a means of initiating the DNA damage response. Disclosures: No relevant conflicts of interest to declare.

Hypotonicity increases transcription, expression, and action ofEgr-1in murine renal medullary mIMCD3 cells

1997 ◽  
Vol 273 (5) ◽  
pp. F837-F842 ◽  
Author(s):  
Zheng Zhang ◽  
David M. Cohen
Keyword(s):  
Dna Binding ◽  
Reporter Gene ◽  
Collecting Duct ◽  
Consensus Sequence ◽  
Luciferase Reporter ◽  
Northern Analysis ◽  
Luciferase Reporter Gene ◽  
Hypotonic Stress ◽  
Gene Assay ◽  
In Cells

In cells of the murine renal inner medullary collecting duct (mIMCD3) cell line, acute hypotonic shock (50% dilution of medium with sterile water but not with sterile 150 mM NaCl) increased Egr-1 mRNA abundance 2.5-fold at 6 h, as determined by Northern analysis. This increase was accompanied by increased Egr-1 transcription, as quantitated by luciferase reporter gene assay. Increased transcription was dose dependent, additive with other Egr-1 transcriptional activators, and occurred in the absence of overt cytotoxicy, as quantitated via a fluorometric viability assay. In addition, hypotonic stress increased Egr-1 protein abundance, which was accompanied by augmented Egr-1-specific DNA binding ability, as measured via electrophoretic mobility shift assay. Increased DNA binding was further associated with increased transactivation by Egr-1, demonstrated through transient transfection of mIMCD3 cells with a luciferase reporter gene driven by tandem repeats of the Egr-1 DNA consensus sequence. Taken together, these data indicate that hypotonic stress activates Egr-1 transcription, translation, DNA binding, and transactivation in renal medullary cells. This phenomenon might play a role in the acquisition of the adaptive phenotype in response to hypotonic stress in cells of the renal medulla in vivo.

scholarly journals miR-27b promotes type II collagen expression by targetting peroxisome proliferator-activated receptor-γ2 during rat articular chondrocyte differentiation

2018 ◽  
Vol 38 (1) ◽  
Author(s):  
Jinying Xu ◽  
Shuang Lv ◽  
Yi Hou ◽  
Kan Xu ◽  
Dongjie Sun ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Type Ii Collagen ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Dependent Manner ◽  
Type Ii ◽  
Peroxisome Proliferator Activated Receptor ◽  
Protein Levels ◽  
Pparγ Agonist ◽  
Gene Assay

MicroRNAs (miRNAs) play an essential role in articular cartilage development and growth. However, the exact mechanisms involved in this process remain unknown. In the present study, we investigated the biological functions of miR-27b during hypertrophic differentiation of rat articular chondrocytes. Based on in situ hybridization and immunohistochemistry, we report that miR-27b expression is reduced in the hypertrophic zone of articular cartilage, but expression of peroxisome proliferator-activated receptor γ (Pparγ) is increased. Dual-luciferase reporter gene assay and Western blot analysis demonstrated that Pparγ2 is a target of miR-27b. Overexpression of miR-27b inhibited expression of Pparγ2, as well as type X collagen (Col10a1) and matrix metalloproteinase 13 (Mmp13), while significantly promoting the expression of Sex-determining Region-box 9 (Sox9) and type II collagen (Col2a1) at both the mRNA and protein levels. Rosiglitazone, a Pparγ agonist, suppressed Col2a1 expression, while promoting expression of runt-related transcription factor 2 (Runx2) and Col10a1 in a concentration-dependent manner. siRNA-mediated knockdown of Pparγ2 caused an increase in protein levels of Col2a1. The present study demonstrates that miR-27b regulates chondrocyte hypertrophy in part by targetting Pparγ2, and that miR-27b may have important therapeutic implications in cartilage diseases.

The Natural Flavonoid Naringenin Inhibits the Cell Growth of WilmsTumorinChildren by Suppressing TLR4/NF-κB Signaling

2020 ◽  
Vol 20 ◽  
Author(s):  
Hongtao Li ◽  
Peng Chen ◽  
Lei Chen ◽  
Xinning Wang
Keyword(s):  
Cell Growth ◽  
Western Blot ◽  
Wilms Tumor ◽  
Critical Role ◽  
Time Dependent ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Tlr4 Expression ◽  
Protein Levels

Background: Nuclear factor kappa B (NF-κB) is usually activated in Wilms tumor (WT) cells and plays a critical role in WT development. Objective: The study purpose was to screen a NF-κB inhibitor from natural product library and explore its effects on WT development. Methods: Luciferase assay was employed to assess the effects of natural chemical son NF-κB activity. CCK-8 assay was conducted to assess cell growth in response to naringenin. WT xenograft model was established to analyze the effect of naringenin in vivo. Quantitative real-time PCR and Western blot were performed to examine the mRNA and protein levels of relative genes, respectively. Results: Naringenin displayed significant inhibitory effect on NF-κB activation in SK-NEP-1 cells. In SK-NEP-1 and G-401 cells, naringenin inhibited p65 phosphorylation. Moreover, naringenin suppressed TNF-α-induced p65 phosphorylation in WT cells. Naringenin inhibited TLR4 expression at both mRNA and protein levels in WT cells. CCK-8 staining showed that naringenin inhibited cell growth of the two above WT cells in dose-and time-dependent manner, whereas Toll-like receptor 4 (TLR4) over expression partially reversed the above phenomena. Besides, naringenin suppressed WT tumor growth in dose-and time-dependent manner in vivo. Western blot found that naringenin inhibited TLR4 expression and p65 phosphorylation in WT xenograft tumors. Conclusion: Naringenin inhibits WT development viasuppressing TLR4/NF-κB signaling

Platelet-Rich and Platelet-Poor Plasma Might Play Supportive Roles in Cancer Cell Culture: A Replacement for Fetal Bovine Serum?

2021 ◽  
Vol 21 ◽  
Author(s):  
Mehdi Talebi ◽  
Mousa Vatanmakanian ◽  
Ali Mirzaei ◽  
Yaghoub Barfar ◽  
Maryam Hemmatzadeh ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Growth ◽  
Cancer Cell ◽  
Human Cancer ◽  
High Price ◽  
Dependent Manner ◽  
Platelet Poor Plasma ◽  
Protein Levels ◽  
Healthy Donors ◽  
Regulatory Functions

Background: Platelet-rich (PRP) and Platelet-poor plasma (PPP) are widely used in research and clinical platforms mainly due to their capacities to enhance cell growth. Although short half-life (5 days) and the high price of platelet products pose challenges regarding their usage, they maintain the growth regulatory functions for weeks. Thus, we aimed to assess the supplementary values of these products in human CCRF-CEM cancer cells. Mechanistically, we also checked if the PRP/PPP treatment enhances YKL-40 expression as a known protein regulating cell growth. Methods: The PRP/PPP was prepared from healthy donors using manual stepwise centrifugation and phase separation. The viability of the cells treated with gradient PRP/PPP concentrations (2, 5, 10, and 15%) was measured by the MTT assay. The YKL-40 mRNA and protein levels were assessed using qRT-PCR and western blotting. The data were compared to FBS-treated cells. Result: Our findings revealed that the cells treated by PRP/PPP not only were morphologically comparable to those treated by FBS but also, they showed greater viability at the concentrations of 10 and 15%. Moreover, it was shown that PRP/PPP induce cell culture support, at least in part, via inducing YKL-40 expression at both mRNA and protein levels in a time- and dose-dependent manner. Conclusion: Collectively, by showing cell culture support comparable to FBS, the PRP/PPP might be used as good candidates to supplement the cancer cell culture and overcome concerns regarding the use of FBS as a non-human source in human cancer research.

Sign in / Sign up

Export Citation Format

Share Document