n-6 Linoleic Acid Induces Epigenetics Alterations Associated with Colonic Inflammation and Cancer
"> Figure 1
<p>An <span class="html-italic">n</span>-6HFD (diet high in <span class="html-italic">n</span>-6 fatty acids) increases body weight and increases the expression of farnesoid-X-receptor (<span class="html-italic">Fxr</span>) in mouse colonic mucosa. (<b>A</b>) Weaned C57/BL6 male mice (<span class="html-italic">n</span> = 6) were assigned to a control diet containing 13% energy (13%E, 5% by weight safflower oil (SO), 76% linoleic acid (LA)) or an <span class="html-italic">n</span>-6HFD containing 44 energy (44%E, 22% by weight SO) for 6 weeks. Bars represent means ± standard error of the mean (SEM) of quantitation (fold change of control) of (<b>B</b>) <span class="html-italic">Fxr</span> and peroxisome proliferator-activated receptorγ1 (<span class="html-italic">Pparγ1</span>), (<b>C</b>) ileal bile acid-binding protein (<span class="html-italic">Ibabp</span>), and (<b>D</b>) small heterodimer protein (<span class="html-italic">Shp</span>) mRNA corrected for glyceraldehyde dehydrogenase phosphate (<span class="html-italic">Gapdh</span>) mRNA as an internal standard. Means ± SEM (standard error of the mean) with an asterisk (*) differ (<span class="html-italic">p</span> < 0.05).</p> "> Figure 2
<p>An <span class="html-italic">n</span>-6HFD induces <span class="html-italic">Fxr</span> CpG demethylation in mouse colonic mucosa. (<b>A</b>) Organization of the mouse <span class="html-italic">Fxr</span> gene. The top arrows indicate transcription start sites (+1) on exon-1 and exon-3. The bottom arrows indicate positions of oligonucleotides (−54/+416) around exon-3 used for CpG methylation studies [<a href="#B25-nutrients-11-00171" class="html-bibr">25</a>]. (<b>B</b>) PCR bands amplified from bisulfonated genomic DNA obtained from proximal colonic mucosa with mouse <span class="html-italic">Fxr</span>- and <span class="html-italic">β-actin</span>-methylation (M)-specific primers. MC = methylation control. (<b>C</b>) Quantitation (fold-change/control) of <span class="html-italic">Fxr</span> promoter methylation status with control (<span class="html-italic">n</span> = 5) and <span class="html-italic">n</span>-6HFD (<span class="html-italic">n</span> = 6). Means ± SEM with an asterisk differ (<span class="html-italic">p</span> < 0.05).</p> "> Figure 3
<p>An <span class="html-italic">n</span>-6HFD induces <span class="html-italic">Ptsg-2</span> (prostaglandin-endoperoxide synthase-2) CpG demethylation in mouse colonic mucosa. (<b>A</b>) The bars represent means ± SEM of quantitation (fold change of control) of <span class="html-italic">Cox-2</span> mRNA corrected for <span class="html-italic">Gapdh</span> mRNA as an internal standard. Means ± SEM with an asterisk differ (<span class="html-italic">p</span> < 0.05). (<b>B</b>) PCR bands amplified from bisulfonated genomic DNA obtained from proximal colonic mucosa with mouse <span class="html-italic">Ptsg-2</span>- and <span class="html-italic">β-actin</span>-methylation (M)-specific primers. MC = methylation control. (<b>C</b>) Quantitation (fold-change/control) of <span class="html-italic">Ptsg-2</span> promoter methylation status with control (<span class="html-italic">n</span> = 5) and <span class="html-italic">n</span>-6HFD (<span class="html-italic">n</span> = 6).</p> "> Figure 4
<p>An <span class="html-italic">n</span>-6HFD induces COX-2 expression in mouse colonic mucosa. (<b>A</b>) The bands are representative immunocomplexes for COX-2 and internal control β-ACTIN in colonic mucosa of mice fed a control or an <span class="html-italic">n</span>-6HFD. (<b>B</b>) The bands are representative control immunocomplexes for COX-2 and internal control β-ACTIN from two separate experiments performed in triplicate in cell lysates of human fetal cells (FHC) cultured in control DMEM (Dulbecco’s Modified Eagle’s Medium) or DMEM plus LA (linoleic acid) (75 μM) for 72 h.</p> "> Figure 5
<p>An <span class="html-italic">n</span>-6HFD induces <span class="html-italic">Apc</span> CpG hypermethylation in mouse colonic mucosa. (<b>A</b>) Quantitation (fold-change/control) of <span class="html-italic">Apc</span> promoter methylation status with control (<span class="html-italic">n</span> = 5) and <span class="html-italic">n</span>-6HFD (<span class="html-italic">n</span> = 6). (<b>B</b>) and (<b>C</b>) The bars represent respectively means ± SEM of quantitation (fold change of control) of <span class="html-italic">Apc</span> and <span class="html-italic">Ccnd1</span> mRNA corrected for <span class="html-italic">Gapdh</span> mRNA as an internal standard. Means ± SEM with an asterisk differ (<span class="html-italic">p</span> < 0.05). (<b>D</b>) The bands are representative immunocomplexes performed in duplicate for c-JUN, COX-2, histone deacetylase-1 (HDAC-1), acetylated histone 3 (ACH3), and internal control β-ACTIN in colonic mucosa of mice fed a control or <span class="html-italic">n</span>-6HFD.</p> "> Figure 6
<p>Proposed epigenetic model of colonic inflammation and carcinogenesis associated with long-term exposure to a diet high in <span class="html-italic">n</span>-6 fatty acids (<span class="html-italic">n</span>-6HFD). Chronic exposure to an <span class="html-italic">n</span>-6HFD impairs adenomatous polyposis Coli (<span class="html-italic">APC</span>) expression through CpG hypermethylation (CpG-CH<sub>3</sub>). This favors the activation of downstream targets of the β-catenin/Wnt pathway, supporting increased expression of genes involved in inflammation (c-JUN, <span class="html-italic">PTSG-2</span>) and proliferation such as cyclin D1 (CCND1). The factors in dark grey boxes refer to changes in expression from the current study. The involvement of β-catenin and upregulation of cyclooxygenase-2 (COX-2) by small heterodimer protein (SHP) are suggested based on published studies mentioned in the Discussion. FXR = farnesoid-X-receptor; IBABP = ileal bile acid-binding protein.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animal Models
2.2. Cell Lines and Reagents
2.3. Real-Time PCR
2.4. Genomic DNA Methylation
2.5. Statistical Analysis
3. Results
3.1. n-6HFD Induces Fxr Gene Promoter Hypomethylation and Expression in Proximal Colonic Mucosa
3.2. n-6HFD Induces Ptsg-2 Gene Promoter Hypomethylation and Expression in Proximal Colonic Mucosa
3.3. n-6HFD Lowers the Expression of Apc and Activates Downstream Targets for the β-Catenin/Wnt Pathway
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Diet | Control | n-6HFD | ||
---|---|---|---|---|
Formula | (g/Kg) | (g/Kg) | ||
Casein | 200.0 | 240.0 | ||
L-Cystine | 3.0 | 3.6 | ||
Corn Starch | 397.5 | 199.4 | ||
Maltodextrin | 132.0 | 150.0 | ||
Sucrose | 120.0 | 80.0 | ||
Safflower Oil | 50.0 | 220.0 | ||
Cellulose | 50.0 | 50.0 | ||
Mineral Mix, AIN-93G-MX(94046) | 35.0 | 42.0 | ||
Mineral Mix, AIN-93-VX(94047) | 10.0 | 12.0 | ||
Choline Bitartrate | 2.5 | 3.0 | ||
TBHQ, Antioxidant | 0.01 | 0.045 | ||
Nutrient Composition | (% Weight) | (% Kcal) | (% Weight) | (% Kcal) |
Protein | 17.7 | 19.3 | 21.2 | 18.7 |
Carbohydrate | 62.1 | 67.9 | 42.3 | 37.3 |
Fat | 5.2 | 12.8 | 22.2 | 44.0 |
Energy (Kcal/g) | 3.7 | 4.5 |
Target | Primer Sequence a |
---|---|
mRNA: | |
Fxr | F: TTAGTCTTCACCACAGCCACC |
R: ACCTGTATACATACATTCAGCCAAC | |
Apc | F: CTGAGCCTGGATGAGCCATT |
R: GTGAGTCCAAGGCGAACGTC | |
Pparγ1 | F: GTGAGACCAACAGCCTGACG |
R: ACAGACTCGGCACTCAATGG | |
Cox-2 | F: GAAGTCTTTGGTCTGGTGCCT |
R: GCTCCTGCTTGAGTATGTCG | |
Gapdh | F: CACTTGAAGGGTGGAGCCAA |
R: AGTGATGGCATGGACTGTGG | |
Ibabp | F: CAGGAGACGTGATTGAAAGGG |
R: GCCCCCAGAGTAAGACTGGG | |
Shp | F: GTACCTGAAGGGCACGATCC |
R: AGCCTCCTGTTGCAGGTGT | |
Ccnd1 | F: CTAAACAAGCACCCCCTCCA |
R: GGTAACAGGGCTGTAGGCAC | |
Methylation-specific: | |
Fxr | F: CGTTTAGCGATGGGGTTAATTAG |
R: CGTCTTCTTTACTTATCTAAACCTCCTT | |
Apc | F: GAGTGTGGTTGTCGGAAATTC |
R: CAAAAAAACGTACATAAAAAACGCT | |
Ptsg-2 | F: TTTTAGTTAGGATTTTAGATTTCGG |
R: ATAATACCAAAAAAACTACACCGC | |
β-Actin | F: AATAGTTATTTTAAGTATTTATGAAATAAG |
R: TAACTACCTCAACACCTCAAC |
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Romagnolo, D.F.; Donovan, M.G.; Doetschman, T.C.; Selmin, O.I. n-6 Linoleic Acid Induces Epigenetics Alterations Associated with Colonic Inflammation and Cancer. Nutrients 2019, 11, 171. https://doi.org/10.3390/nu11010171
Romagnolo DF, Donovan MG, Doetschman TC, Selmin OI. n-6 Linoleic Acid Induces Epigenetics Alterations Associated with Colonic Inflammation and Cancer. Nutrients. 2019; 11(1):171. https://doi.org/10.3390/nu11010171
Chicago/Turabian StyleRomagnolo, Donato F., Micah G. Donovan, Tom C. Doetschman, and Ornella I. Selmin. 2019. "n-6 Linoleic Acid Induces Epigenetics Alterations Associated with Colonic Inflammation and Cancer" Nutrients 11, no. 1: 171. https://doi.org/10.3390/nu11010171