Identification of Potential Methylation Regions of the Smad4 mRNA and Determining Primer Sequences for  MS-PCR with the ‘Methprimer’ Program

Metin Budak; Ugur Ozkan; Mustafa Yildiz

doi:doi:10.11648/j.ijgg.20190703.13

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Identification of Potential Methylation Regions of the Smad4 mRNA and Determining Primer Sequences for MS-PCR with the ‘Methprimer’ Program

Metin Budak, Ugur Ozkan, Mustafa Yildiz

Received: 16 July 2019 Accepted: 12 August 2019 Published: 20 August 2019

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Abstract

SMAD4 is a member of an intracellular signaling pathway protein family that is widely expressed in human tissues. This protein is responsible for carrying a chemical signal from the cell membrane to the nucleus. Since reduced SMAD4 expression leads to several tumors and neural disease, it is important to elucidate the mechanisms affecting the expression of this protein. Methylation is among the major factors that affect the expression of the SMAD4 gene. While methylation of the promoter and non-coding exons of SMAD4 gene appear to affect expression, there is no information regarding the other regions of this gene in this regard. Furthermore, cytosine methylation in mRNA is also important in gene activity. For this reason, the demonstration of possible cytosine methylation in mRNA of the SMAD4 gene may be important in understanding gene activity. In this study, we aimed to determine the potential methylation regions in the exons corresponding to SMAD4 protein generation which have not been investigated before. In order to do this, we used the MethPrimer program and identified 25 single CpG sequences and a double CpGpCpG across the exons as potential methylation regions. In addition, 5 pairs of methylated/unmethylated primer sequences were designed with the same program. The study results have shown the presence of potential methylation sequences that are candidates to affect SMAD4 gene expression.

DOI	10.11648/j.ijgg.20190703.13
Published in	International Journal of Genetics and Genomics (Volume 7, Issue 3, September 2019)
Page(s)	55-59
Creative Commons	This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.
Copyright	Copyright © The Author(s), 2024. Published by Science Publishing Group

Keywords

Smad4 Protein, Methylation, mRNA, PCR

References

[1]	S. Roth, P. Laiho, R. Salovaara, V. Launonen, L. A. Aaltonen, (2000). No SMAD4 hypermethylation in colorectal cancer, British journal of cancer 83 (8); 1015.
[2]	L. P. Cacheaux, S. Ivens, Y. David, A. J. Lakhter, G. Bar-Klein, M. Shapira, U. Heinemann, A. Friedman, D. Kaufer, (2009). Transcriptome profiling reveals TGF-β signaling involvement in epileptogenesis, Journal of Neuroscience 29 (28); 8927-8935.
[3]	O. Legendre, A. Sookdeo, D. A. Foster, (2014). BxPC3 pancreatic cancer cells express a truncated Smad4 protein upon PI3K and mTOR inhibition, Oncology letters 7 (4); 1165-1168.
[4]	L. Fagerberg, B. M. Hallström, P. Oksvold, C. Kampf, D. Djureinovic, J. Odeberg, M. Habuka, S. Tahmasebpoor, A. Danielsson, K. Edlund, (2014). Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics, Molecular & Cellular Proteomics 13 (2); 397-406.
[5]	R. Jaenisch, A. Bird, (2003). Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals, Nature genetics 33: 245.
[6]	B. M. Turner, (2000). Histone acetylation and an epigenetic code, Bioessays 22 (9); 836-845.
[7]	B. H. Ramsahoye, D. Biniszkiewicz, F. Lyko, V. Clark, A. P. Bird, R. Jaenisch, (2000). Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a, Proceedings of the National Academy of Sciences 97 (10); 5237-5242.
[8]	T. Pollex, K. Hanna, M. Schaefer, (2010). Detection of cytosine methylation in RNA using bisulfite sequencing, Cold Spring Harbor Protocols 2010 (10); pdb. prot5505.
[9]	M. Schaefer, T. Pollex, K. Hanna, F. Lyko, (2008). RNA cytosine methylation analysis by bisulfite sequencing, Nucleic acids research 37 (2); e12-e12.
[10]	F. Tuorto, R. Liebers, T. Musch, M. Schaefer, S. Hofmann, S. Kellner, M. Frye, M. Helm, G. Stoecklin, F. Lyko, (2012). RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis, Nature Structural and Molecular Biology 19 (9); 900.
[11]	L. -C. Li, R. Dahiya, (2002). MethPrimer: designing primers for methylation PCRs, Bioinformatics 18 (11); 1427-1431.
[12]	S. A. Hahn, M. Schutte, A. S. Hoque, C. A. Moskaluk, L. T. Da Costa, E. Rozenblum, C. L. Weinstein, A. Fischer, C. J. Yeo, R. H. Hruban, (1996). DPC4, a candidate tumor suppressor gene at human chromosome 18q21. 1, Science 271 (5247); 350-353.
[13]	M. Rolando, L. Gomez‐Valero, C. Buchrieser, (2015). Bacterial remodelling of the host epigenome: functional role and evolution of effectors methylating host histones, Cellular microbiology 17 (8); 1098-1107.
[14]	L. -H. Wang, S. -H. Kim, J. H. Lee, Y. -L. Choi, Y. C. Kim, T. S. Park, Y. -C. Hong, C. -F. Wu, Y. K. Shin, (2007). Inactivation of SMAD4 tumor suppressor gene during gastric carcinoma progression, Clinical cancer research 13 (1); 102-110.
[15]	F. Brenet, M. Moh, P. Funk, E. Feierstein, A. J. Viale, N. D. Socci, J. M. Scandura, (2011). DNA methylation of the first exon is tightly linked to transcriptional silencing, PloS one 6 (1) e14524.
[16]	F. Yang, H. Jin, B. Que, Y. Chao, H. Zhang, X. Ying, Z. Zhou, Z. Yuan, J. Su, B. Wu, (2019). Dynamic m 6 A mRNA methylation reveals the role of METTL3-m 6 A-CDCP1 signaling axis in chemical carcinogenesis, Oncogene 38 (24); 4755.
[17]	M. Engel, A. Chen, (2018). The emerging role of mRNA methylation in normal and pathological behavior, Genes, Brain and Behavior 17 (3); e12428.
[18]	N. A. Gkatza, C. Castro, R. F. Harvey, M. Heiß, M. C. Popis, S. Blanco, S. Bornelöv, A. A. Sajini, J. G. Gleeson, J. L. Griffin, (2019). Cytosine-5 RNA methylation links protein synthesis to cell metabolism, PLoS biology 17 (6); e3000297.
[19]	Y. Motorin, F. Lyko, M. Helm, (2009). 5-methylcytosine in RNA: detection, enzymatic formation and biological functions, Nucleic acids research 38 (5); 1415-1430.
[20]	M. Budak, (2018). Logic of Epigenetics and Investigation of Potential Gene Regions, Epigenetics, Intech Open.
[21]	O. Yalcin, M. Budak, (2017). Un-methylation of the survivin gene has no effect on immunohistochemical expression of survivin protein in lung cancer patients with squamous cell carcinoma, Bratislavske lekarske listy 118 (3); 160-163.

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APA Style

Metin Budak, Ugur Ozkan, Mustafa Yildiz. (2019). Identification of Potential Methylation Regions of the Smad4 mRNA and Determining Primer Sequences for MS-PCR with the ‘Methprimer’ Program. International Journal of Genetics and Genomics, 7(3), 55-59. https://doi.org/10.11648/j.ijgg.20190703.13

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ACS Style

Metin Budak; Ugur Ozkan; Mustafa Yildiz. Identification of Potential Methylation Regions of the Smad4 mRNA and Determining Primer Sequences for MS-PCR with the ‘Methprimer’ Program. Int. J. Genet. Genomics 2019, 7(3), 55-59. doi: 10.11648/j.ijgg.20190703.13

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AMA Style

Metin Budak, Ugur Ozkan, Mustafa Yildiz. Identification of Potential Methylation Regions of the Smad4 mRNA and Determining Primer Sequences for MS-PCR with the ‘Methprimer’ Program. Int J Genet Genomics. 2019;7(3):55-59. doi: 10.11648/j.ijgg.20190703.13

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@article{10.11648/j.ijgg.20190703.13,
  author = {Metin Budak and Ugur Ozkan and Mustafa Yildiz},
  title = {Identification of Potential Methylation Regions of the Smad4 mRNA and Determining Primer Sequences for  MS-PCR with the ‘Methprimer’ Program},
  journal = {International Journal of Genetics and Genomics},
  volume = {7},
  number = {3},
  pages = {55-59},
  doi = {10.11648/j.ijgg.20190703.13},
  url = {https://doi.org/10.11648/j.ijgg.20190703.13},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijgg.20190703.13},
  abstract = {SMAD4 is a member of an intracellular signaling pathway protein family that is widely expressed in human tissues. This protein is responsible for carrying a chemical signal from the cell membrane to the nucleus. Since reduced SMAD4 expression leads to several tumors and neural disease, it is important to elucidate the mechanisms affecting the expression of this protein. Methylation is among the major factors that affect the expression of the SMAD4 gene. While methylation of the promoter and non-coding exons of SMAD4 gene appear to affect expression, there is no information regarding the other regions of this gene in this regard. Furthermore, cytosine methylation in mRNA is also important in gene activity. For this reason, the demonstration of possible cytosine methylation in mRNA of the SMAD4 gene may be important in understanding gene activity. In this study, we aimed to determine the potential methylation regions in the exons corresponding to SMAD4 protein generation which have not been investigated before. In order to do this, we used the MethPrimer program and identified 25 single CpG sequences and a double CpGpCpG across the exons as potential methylation regions. In addition, 5 pairs of methylated/unmethylated primer sequences were designed with the same program. The study results have shown the presence of potential methylation sequences that are candidates to affect SMAD4 gene expression.},
 year = {2019}
}

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TY - JOUR
T1 - Identification of Potential Methylation Regions of the Smad4 mRNA and Determining Primer Sequences for MS-PCR with the ‘Methprimer’ Program
AU - Metin Budak
AU - Ugur Ozkan
AU - Mustafa Yildiz
Y1 - 2019/08/20
PY - 2019
N1 - https://doi.org/10.11648/j.ijgg.20190703.13
DO - 10.11648/j.ijgg.20190703.13
T2 - International Journal of Genetics and Genomics
JF - International Journal of Genetics and Genomics
JO - International Journal of Genetics and Genomics
SP - 55
EP - 59
PB - Science Publishing Group
SN - 2376-7359
UR - https://doi.org/10.11648/j.ijgg.20190703.13
AB - SMAD4 is a member of an intracellular signaling pathway protein family that is widely expressed in human tissues. This protein is responsible for carrying a chemical signal from the cell membrane to the nucleus. Since reduced SMAD4 expression leads to several tumors and neural disease, it is important to elucidate the mechanisms affecting the expression of this protein. Methylation is among the major factors that affect the expression of the SMAD4 gene. While methylation of the promoter and non-coding exons of SMAD4 gene appear to affect expression, there is no information regarding the other regions of this gene in this regard. Furthermore, cytosine methylation in mRNA is also important in gene activity. For this reason, the demonstration of possible cytosine methylation in mRNA of the SMAD4 gene may be important in understanding gene activity. In this study, we aimed to determine the potential methylation regions in the exons corresponding to SMAD4 protein generation which have not been investigated before. In order to do this, we used the MethPrimer program and identified 25 single CpG sequences and a double CpGpCpG across the exons as potential methylation regions. In addition, 5 pairs of methylated/unmethylated primer sequences were designed with the same program. The study results have shown the presence of potential methylation sequences that are candidates to affect SMAD4 gene expression.
VL - 7
IS - 3
ER -

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Author Information

Metin Budak

Department of Biophysics, Faculty of Medicine, Trakya University, Edirne, Turkey; Mirko Tos Ear and Hearing Research Center, Trakya University, Edirne, Turkey
Ugur Ozkan

Department of Cardiology, Edirne Sultan 1. Murat State Hospital, Edirne, Turkey
Mustafa Yildiz

Department of Biophysics, Faculty of Medicine, Trakya University, Edirne, Turkey

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APA Style

Metin Budak, Ugur Ozkan, Mustafa Yildiz. (2019). Identification of Potential Methylation Regions of the Smad4 mRNA and Determining Primer Sequences for MS-PCR with the ‘Methprimer’ Program. International Journal of Genetics and Genomics, 7(3), 55-59. https://doi.org/10.11648/j.ijgg.20190703.13

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ACS Style

Metin Budak; Ugur Ozkan; Mustafa Yildiz. Identification of Potential Methylation Regions of the Smad4 mRNA and Determining Primer Sequences for MS-PCR with the ‘Methprimer’ Program. Int. J. Genet. Genomics 2019, 7(3), 55-59. doi: 10.11648/j.ijgg.20190703.13

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AMA Style

Metin Budak, Ugur Ozkan, Mustafa Yildiz. Identification of Potential Methylation Regions of the Smad4 mRNA and Determining Primer Sequences for MS-PCR with the ‘Methprimer’ Program. Int J Genet Genomics. 2019;7(3):55-59. doi: 10.11648/j.ijgg.20190703.13

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@article{10.11648/j.ijgg.20190703.13,
  author = {Metin Budak and Ugur Ozkan and Mustafa Yildiz},
  title = {Identification of Potential Methylation Regions of the Smad4 mRNA and Determining Primer Sequences for  MS-PCR with the ‘Methprimer’ Program},
  journal = {International Journal of Genetics and Genomics},
  volume = {7},
  number = {3},
  pages = {55-59},
  doi = {10.11648/j.ijgg.20190703.13},
  url = {https://doi.org/10.11648/j.ijgg.20190703.13},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijgg.20190703.13},
  abstract = {SMAD4 is a member of an intracellular signaling pathway protein family that is widely expressed in human tissues. This protein is responsible for carrying a chemical signal from the cell membrane to the nucleus. Since reduced SMAD4 expression leads to several tumors and neural disease, it is important to elucidate the mechanisms affecting the expression of this protein. Methylation is among the major factors that affect the expression of the SMAD4 gene. While methylation of the promoter and non-coding exons of SMAD4 gene appear to affect expression, there is no information regarding the other regions of this gene in this regard. Furthermore, cytosine methylation in mRNA is also important in gene activity. For this reason, the demonstration of possible cytosine methylation in mRNA of the SMAD4 gene may be important in understanding gene activity. In this study, we aimed to determine the potential methylation regions in the exons corresponding to SMAD4 protein generation which have not been investigated before. In order to do this, we used the MethPrimer program and identified 25 single CpG sequences and a double CpGpCpG across the exons as potential methylation regions. In addition, 5 pairs of methylated/unmethylated primer sequences were designed with the same program. The study results have shown the presence of potential methylation sequences that are candidates to affect SMAD4 gene expression.},
 year = {2019}
}

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