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DNA methylation in the placenta and in cancerwith special reference to folate transporting genes
Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

DNA methylation is an epigenetic mechanism that regulates the gene transcription. Folate is used in cellular synthesis of methyl groups, nucleic acids and amino acids. In complex diseases like cancer and neural tube defects (NTD), various genetic and epigenetic alterations can be found that disrupt the normal cell function. The main goals of this thesis were to analyze whether the genes responsible for the folate transport (FOLR1, PCFT, and RFC1) could be regulated by DNA methylation in placenta, blood leukocytes and colorectal cancer. We also addressed the genome-wide DNA methylation changes in colorectal cancer andcervical cancer.We found that changes in the methylated fraction of the RFC1 gene were dependent on the RFC1 80G>A polymorphism in placental specimens with NTDs and blood leukocytes from subjects with high homocysteine (Paper I). In colorectal cancer, the greatest difference in DNA methylation was observed in the RFC1 gene and was related to a lower protein expression (Paper II).In Paper III and IV we studied the DNA methylated fraction using a high-density array. Paper III was focused on genes in the DNA repair pathway and frequently mutated in colorectal cancer. We found that aberrant methylation in the DNA mismatch repair genes was not a frequent event in colorectal cancer and we identified five candidate biomarker genes in colorectal cancer, among them the GPC6 and DCLRE1C genes. In Paper IV, we found hypomethylation of genes involved in the immune system in cervical cancer specimens compared to healthy cervical scrapes and we identified twenty four candidate genes for further evaluation ofclinical value.In conclusion, the work of this thesis filled a relevant knowledge gap regarding the role of differential methylation of the folate transport genes in NTD and colorectal cancer. This thesis work also provided insights into the functional role of DNA methylation in cancer specific pathways and identified potential novel biomarker genes.

Place, publisher, year, edition, pages
Örebro: Örebro university , 2014. , 67 p.
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 100
Keyword [en]
DNA methylation, CRC, placenta, cervix, leukocytes, T-DMRs, folate, array, expression
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biomedicine
Identifiers
URN: urn:nbn:se:oru:diva-32599ISBN: 978-91-7668-986-8 (print)OAI: oai:DiVA.org:oru-32599DiVA: diva2:668943
Public defence
2014-01-31, Wilandersalen, Universitetssjukhuset, S. Grev Rosengatan, 703 62 Örebro, 09:15 (English)
Opponent
Supervisors
Available from: 2013-12-02 Created: 2013-12-02 Last updated: 2017-10-17Bibliographically approved
List of papers
1. Epigenetic alterations in folate transport genes in placental tissue from fetuses with neural tube defects and in leukocytes from subjects with hyperhomocysteinemia
Open this publication in new window or tab >>Epigenetic alterations in folate transport genes in placental tissue from fetuses with neural tube defects and in leukocytes from subjects with hyperhomocysteinemia
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2013 (English)In: Epigenetics, ISSN 1559-2294, E-ISSN 1559-2308, Vol. 8, no 3, 303-316 p.Article in journal (Refereed) Published
Abstract [en]

The objectives of this study were to identify tissue-specific differentially methylated regions (T-DMR's) in the folate transport genes in placental tissue compared with leukocytes, and from placental tissues obtained from normal infants or with neural tube defects (NTDs). Using pyrosequencing, we developed methylation assays for the CpG islands (CGIs) and the CGI shore regions of the folate receptor a (FOLR1), proton-coupled folate transporter (PCFT) and reduced folate carrier 1 (RFC1) genes. The T-DMRs differed in location for each gene and the difference in methylation ranged between 2 and 54%. A higher T-DMR methylated fraction was associated with a lower mRNA level of the FOLR1 and RFC1 genes. Methylation fractions differed according to RFC1 80G > A genotype in the NTD cases and in leukocytes from subjects with high total plasma homocysteine (tHcy). There were no differences in methylated fraction of folate transporter genes between NTD cases and controls. We suggest that T-DMRs participate in the regulation of expression of the FOLR1 and RFC1 genes, that the RFC1 80G > A polymorphism exerts a gene-nutrition interaction on DNA methylation in the RFC1 gene, and that this interaction appears to be most prominent in NTD-affected births and in subjects with high tHcy concentrations.

Keyword
FOLR1, PCFT, RFC1 80G > A, homocysteine, tissue-specific DNA methylation, GpG island, NTD
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-35872 (URN)10.4161/epi.23988 (DOI)000315923300008 ()
Note

Funding agencies:

Lions cancerfond

Nyckelfonden

Orebro lans landsting

NIH Grant numbers: HD067244 ES021390

State Key Development Program for Basic Research, People's Republic of China Grant numbers: 2007CB5119001 

Available from: 2014-08-07 Created: 2014-08-07 Last updated: 2017-10-18Bibliographically approved
2. DNA methylation and expression of the folate transporting genes in colorectal cancer
Open this publication in new window or tab >>DNA methylation and expression of the folate transporting genes in colorectal cancer
(English)Manuscript (preprint) (Other academic)
Abstract [en]

This study investigated the DNA methylation pattern and protein expression of the FOLR1, PCFT, and RFC1 genes in colorectal cancer (CRC) tissue. Our results showed statistically significant differences in the DNA methylated fraction of all three genes at several gene regions, we identified 3 differentially methylated CpG sites in the FOLR1 gene, 5 CpG sites in the PCFT gene, and 6 CpG sites in the RFC1 gene. We observed a pronounced expression of the FRα and RFC proteins in both the cancer and normal tissues, though the proteins were moderately expressed in cancer compared to the high expression in the paired healthy mucosa. The PCFT protein was undetectable or expressed very low in both tissues. Higher methylated fractions of the CpG sites 3-5 in the RFC1 gene were associated with a lower protein expression. When analyzing the association between DNA methylation and tumor characteristics (differentiation, location, primary tumor stage and lymph node involvement) we detected lower methylated fraction of specific CpG sites in the RFC1 gene in CRC located in the distal colon and rectum compared to the proximal colon. In the FOLR1 gene, we found CpG sites with a lower methylated fraction of colorectal cancers with the primary tumor stage 4 (pT4) compared to the pT2 and pT3 stages. Our results did not show association between the RFC and FRα protein expression and tumor stage, TNM classification or tumor location. In conclusion, these data suggest that there is a possible epigenetic regulation by DNA methylation of the RFC1 gene in the colorectal cancer.

Keyword
CpG, T-DMR, reduced folate carrier, CRC, immunohistochemistry
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-35869 (URN)
Available from: 2014-08-07 Created: 2014-08-07 Last updated: 2017-10-17Bibliographically approved
3. DNA methylation changes in genes frequently mutated in sporadic colorectal cancer and in the DNA repair and Wnt/beta-catenin signaling pathway genes
Open this publication in new window or tab >>DNA methylation changes in genes frequently mutated in sporadic colorectal cancer and in the DNA repair and Wnt/beta-catenin signaling pathway genes
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2014 (English)In: Epigenomics, ISSN 1750-1911, Vol. 6, no 2, 179-191 p.Article in journal (Refereed) Published
Abstract [en]

Aim: The onset and progression of colorectal cancer (CRC) involves a cascade of genetic and/or epigenetic events. The aim of the present study was to address the DNA methylation status of genes relevant in colorectal carcinogenesis and its progression, such as genes frequently mutated in CRC, genes involved in the DNA repair and Wnt signaling pathway.

Material & methods: We analyzed methylation status in totally 160 genes in 12 paired colorectal tumors and adjacent healthy mucosal tissues using the Illumina Infinium Human Methylation 450 BeadChip.

Results: We found significantly aberrant methylation in 23 genes (NEIL1, NEIL3, DCLRE1C, NHEJ1, GTF2H5, CCNH, CTNNB1, DKK2, DKK3, FZD5 LRP5, TLE3, WNT2, WNT3A, WNT6, TCF7L1, CASP8, EDNRB1, GPC6, KIAA1804, MYO1B, SMAD2 and TTN). External validation by mRNA expression showed a good agreement between hypermethylation in cancer and down-regulated mRNA expression of the genes EDNRB1, GPC6 and SMAD2, and between hypomethylation and up-regulated mRNA expression of the CASP8 and DCLRE1C genes.

Conclusion: Aberrant methylation of the DCLRE1C and GPC6 genes are presented here for the first time and are therefore of special interest for further validation as novel candidate biomarker genes in CRC, and merit further validation with specific assays.

Place, publisher, year, edition, pages
Future Medicine, 2014
Keyword
CpG, DNA repair genes, Infinium Human Methylation 450 BeadChip, methylation status, sporadic colorectal cancer, Wnt/beta-catenin signaling pathway
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Medical Genetics
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-35867 (URN)10.2217/EPI.14.7 (DOI)000335684900015 ()24811787 (PubMedID)2-s2.0-84900412412 (Scopus ID)
Note

Funding agencies:

CZ:GA CR GA Grant numbers: P304/11/P715 P304/12/1585

IGA:NT Grant number: 14329

Lions Cancer Foundation

Nyckelfonden

Örebro läns landsting 

Available from: 2014-08-07 Created: 2014-08-07 Last updated: 2017-12-05Bibliographically approved
4. Genome-wide DNA methylation assay reveals novel candidate biomarker genes in cervical cancer
Open this publication in new window or tab >>Genome-wide DNA methylation assay reveals novel candidate biomarker genes in cervical cancer
2013 (English)In: Epigenetics, ISSN 1559-2294, E-ISSN 1559-2308, Vol. 8, no 11, 1213-1225 p.Article in journal (Refereed) Published
Abstract [en]

The oncogenic human papilloma viruses (HPVs) are associated with precancerous cervical lesions and development of cervical cancer. The DNA methylation signatures of the host genome in normal, precancerous and cervical cancer tissue may indicate tissue-specific perturbation in carcinogenesis. The aim of this study was to identify new candidate genes that are differentially methylated in squamous cell carcinoma compared with DNA samples from cervical intraepithelial neoplasia grade 3 (CIN3) and normal cervical scrapes. The Illumina Infinium HumanMethylation450 BeadChip method identifies genome-wide DNA methylation changes in CpG islands, CpG shores and shelves. Our findings showed an extensive differential methylation signature in cervical cancer compared with the CIN3 or normal cervical tissues. The identified candidate biomarker genes for cervical cancer represent several types of mechanisms in the cellular machinery that are epigenetically deregulated by hypermethylation, such as membrane receptors, intracellular signaling and gene transcription. The results also confirm extensive hypomethylation of genes in the immune system in cancer tissues. These insights into the functional role of DNA methylome alterations in cervical cancer could be clinically applicable in diagnostics and prognostics, and may guide the development of new epigenetic therapies.

Keyword
DNA methylation, Infinium Human Methylation 450 BeadChip, HPV, cervical cancer, immune system
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-35866 (URN)10.4161/epi.26346 (DOI)000336517500010 ()
Note

Funding agencies:

Lions Cancer Foundation

Nyckelfonden  

Orebro lans landsting  

Croatian Ministry of Science, Education and Sports, grant number 098-0982464-2510 

Available from: 2014-08-07 Created: 2014-08-07 Last updated: 2017-12-05Bibliographically approved

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