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Sample & Assay Technologies Oxidative Stress and ROS Signaling
� QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
� For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.
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Sample & Assay Technologies Oxidative Stress and ROS Signaling
� Overview� Oxidants & antioxidant in biological
systems
� Antioxidant defense mechanism
� Molecular targets of oxidative stress
� Detection of oxidative stress
� Research Solutions @ QIAGEN� Gene expression
� Epigenetics
� Functional studies
� Application Examples
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Sample & Assay Technologies Oxidative stress and ROS signaling
� Oxidative stress : imbalance between reactive oxygen species (ROS) and antioxidants.
� Under normal conditions, cells are able to balance the production of oxidants and antioxidants. Oxidative stress occurs when cells are subjected to excess levels of ROS or as a result of antioxidant depletion.
Oxidative stress, chronic inflammation, and cancer are closely linked
� Free radicals as a link of oxidative stress to inflammation and cancer
Oxidative stressOxidative stress
Carbonylic adducts Hydro & lipoperoxides Imbalance in DNA repair
Imbalance between pro & anti-apoptotic processes, altered gene expression & mutations
Chronic inflammation
Activation of Inflammatory cells
ROS production
LOXs, COXs, PHGPx iNOS
NFkBCytokines
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Sample & Assay Technologies ROS play multiple roles in the hallmarks of cancers
Review article: “Oxidative Stress, Tumor Microenvironment, and Metabolic Reprogramming: A Diabolic Liaison” International Journal of Cell Biology. 2012
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Sample & Assay Technologies
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� Oxidative stress is tightly linked to
mitochondrial dysfunction, and
mitochondria are both generators of
and targets of ROS
� In respond to cellular stress,
necrosis, apoptosis and autophagy
are activated.
� Mitochondrial turnover is dependent
on autophagy
� Dysregulated redox signaling or
mitochondrial dysfunction affect
autophagic activities
Mitochondria are the targets of ROS
Review article: “Autophagy, mitochondria and oxidative stress: cross-talk and redox signaling” Biochem. J. 2012, 441, 523
Sample & Assay Technologies miRNAs are modulators and targets of oxidative stress
“Inflammation and cancer: interweaving microRNA, free radical, cytokine and p53 pathways” Carcinogenesis, 31(1): 37–49, 2010
miRNAs are non-coding RNA molecules that modulate the stability and/or the translational efficiency of target messenger RNAs
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“Oxidative Stress and MicroRNAs in Vascular Diseases” Int. J. Mol. Sci. 2013, 14
Relevant miRNAs in oxidative stress response
ROS source
miRNAs
upregulated Tissue/organ Source Target Functions
H2O2 miR-200c
miR-141 endothelium
miR-200a myoblasts Human ZEB1 apoptosis
miR-200b
miR-429
H2O2 miR-200c ROS accumulation;
miR-141 miR-200a ovarian Human p38α improved response to
miR-200b adenocarcinomas chemotherapy
miR-429
H2O2 miR-200c primary Mouse Unknown Unknown
hippocampal
neurons
Obesity miR-200c miR-141 Heart Rat S6K1 compensatory/adaptive
mechanisms
Sample & Assay Technologies Detection of intracellular oxidative stress
� Direct approach� Detection of ROS / free radicals
Sample & Assay Technologies Applications of oxidative stress PCR Arrays
Publications Citing the Oxidative Stress PCR Array
• Susceptibility to Inhaled Flame-Generated Ultrafine Soot in Neonatal and Adult Rat Lungs, TOXICOLOGICAL SCIENCES 124(2), 472–486 (2011), Jackie K. W. Chan,* Michelle V, etc.
• Adaptive induction of NF-E2-related factor-2-driven antioxidant genes in endothelial cells in response to hyperglycemia, Am J Physiol Heart Circ Physiol 300: H1133–H1140, 2011, Zoltan Ungvari,1 Lora Bailey-Downs, etc.
• Tryptophan from human milk induces oxidative stress and upregulates the Nrf-2-mediated stress response in human intestinal cell lines. J Nutr. 2011 Aug;141(8):1417-23. Elisia I, Tsopmo A, Friel JK, etc.
• Age-related gene response of human corneal endothelium to oxidative stress and DNA damage. Invest Ophthalmol Vis Sci. 2011 Mar 1;52(3):1641-9. Joyce NC, Harris DL, Zhu CC
• Gene expression analysis of oxidative stress and apoptosis in proton-irradiated rat retina. In Vivo. 2010 Jul-Aug;24(4):425-30. Mao XW, Green LM, Mekonnen T, Lindsey N, Gridley DS
• Increased ANG II sensitivity following recovery from acute kidney injury: role of oxidant stress in skeletal muscle resistance arteries. Am J Physiol Regul Integr Comp Physiol. 2010 Jun;298(6):R1682-91. Phillips SA, etc.
• Effects of spaceflight on innate immune function and antioxidant gene expression. J Appl Physiol. 2009 Jun;106(6):1935-42. Baqai FP, Gridley DS, Slater JM, Luo-Owen X, Stodieck LS, Ferguson V, etc.
• Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defenses. Braz J Med BiolRes. 2005 Jul;38(7):995-1014. Scandalios JG
• Oxidative stress and gene regulation. Free Radic Biol Med. 2000 Feb 1;28(3):463-99. Allen RG, Tresini M
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Sample & Assay Technologies
Transcription factors Nrf2 and Nrf1 bind to the antioxidant response elements (ARE) and regulate genes involved in protecting cells from oxidative damages.
.How to measure the degree of antioxidant response?
Antioxidant Response ReporterCignal Finder Stress & Toxicity 10-pathway reporter Kit
Measure the transcriptional activity of the Nrf2 (NF-E2-related factor 2) and Nrf1 transcription factors. Nrf2 and Nrf1 bind to the antioxidant response elements (AREs) and regulate genes involved in protecting cells from oxidative damage.
Application 2: reporter assays for oxidative stress
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Sample & Assay Technologies
Answer two questions:
1. Whether hyperglycemia activates Nrf2 in endothelial cells?
2. Whether induction of Nrf2-regulated ROS detoxification systems protect
endothelial function in type 2 diabetes?
Experiment procedure: • Treat wild type and Nrf2 knockout mouse with a high-fat diet (HFD) and standard diet
• Use reporter assay to measure Nrf2 activity
Application 2: reporter assays for oxidative stress
• Adaptive activation of the Nrf2/ARE pathway has a critical role in endothelial protection in response
to diabetic condition both in vitro and in vivo.
Application 2: reporter assays for oxidative stress
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Glucose (mmol/L)
Rel
ativ
e N
rf2
indu
ctio
n (f
old
chan
ge)
Glucose (mmol/L)
Rel
ativ
e m
RN
A e
xpre
ssio
n (f
old
chan
ge)
Sample & Assay Technologies
Functional genomics:
�Assess RNA interference phenotypes
�Assess overexpression phenotypes
Functional proteomics:
�Analyze the effects of recombinant protein or peptide treatments
Drug discovery:
�Screen and validate small molecule drug candidates
Applications: Cignal reporter assay kits
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Download a white paper “Cignal Reporter Assay Kit: A high-performance tool for assessing the functions of genes, biologics, and small molecule compounds - (EN)”
� Conclusion: miR-200a regulates the Keap1/Nrf2 pathway in mammary epithelium ; Epigenetic therapy can restore the miR-200a regulation of Keap1 expression, therefore to reactivate Nrf2-dependent antioxidant pathway in breast cancer.
� Measured Nrf2 ARE promoter activity using luciferas e reporter assay
� Epigenetic therapy restored miR-200 expression and reduced Keap1 level
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• miR-200c is a tumor suppressor miRNA, involved in cancers such as breast, ovarian, endometrial, colorectal cancer, and pancreatic cancer
Normal cell: MCF-10A
Bre
ast c
ell l
ine:
MD
A-M
B-2
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Mature miRNAs Fold changes
has-miRNA-141 -869.07
has-miRNA-200c -422.65
has-miRNA-2302c -66.41
has-miRNA-196b -65.95
has-miRNA-143 -40.32
has-miRNA-122 -18.68
has-miRNA-7f -15.07
… …
Sample & Assay Technologies miRNA assays and reagents from QIAGEN
Target Identification Expression
Isolation
Function
miRNA mimics & inhibitors
miScript miRNA PCR Arrays
miRNeasy
miRNAStudies
Feb 6-27, Thursday : 4 part miRNA focused webinar series, monthlyhttps://www2.gotomeeting.com/register/152689794
RT² Profiler™ microRNA Targets PCR Arrays
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Sample & Assay Technologies Oxidative stress and DNA methylation
Stress & Toxicity PathwayFinder DNA Methylation PCR Array