Ineffectiveness of Methylation in Rgulation of VHL, ECAD, and RUNX3 Genes in Erythroid Cells Differentiated by Erythropoietin
Background: Vast variety of intermediate factors including cell cycle regulators, growth factors, transcription factors, and signaling pathways are involved in hematopoietic stem cell (HSC) commitment and differentiation into distinct lineages. VHL, Ecad, and RUNX3 are among these. Epigenetics is currently introduced as a potential mechanism to control the gene regulation. The aim of this study is to reveal the correlation between the expression level and methylation pattern of mentioned genes after in vitro differentiation of cord blood HSCs into erythroid lineage mediated by erythropoietin.
Materials and Methods: After isolation and expansion, the CD34+ cord blood stem cells were divided into two parts. The first part was used to extract the DNA and RNA and the second to differentiate into erythroid lineage. Methylation specific PCR (MSP) and Real-time PCR were used to determine the methylation status and expression levels of the genes, respectively.
Results: Although the significant upregulation observed for VHL and Ecad genes and a down-regulation for RUNX3 gene after differentiation, no remarkable changes were seen in methylation pattern compared with cord blood HSCs by MSP technique.
Conclusion: It is appearing that methylation pattern in promoter region has not an effective role in expression of VHL, Ecad, and RUNX3. Moreover, considering the inability of MSP method to detect subtle differences in methylation level a more sensitive method is needed to distinguish the methylation levels of these genes before and after erythroid differentiation.
Azad M, Kaviani S, Noruzinia M, Mortazavi Y, Mobarra N, Alizadeh S, et al. Gene Expression Status and Methylation Pattern in Promoter of P15INK4b and P16INK4a in Cord Blood CD34 (+) Stem Cells. Iran J Basic Med Sci. 2013;16(7):822-8.
Burns CE, Traver D, Mayhall E, Shepard JL, Zon LI. Hematopoietic stem cell fate is established by the Notch–Runx pathway. Genes & development. 2005;19(19):2331-42.
Wang CQ, Motoda L, Satake M, Ito Y, Taniuchi I, Tergaonkar V, et al. Runx3 deficiency results in myeloproliferative disorder in aged mice. Blood. 2013;122(4):562-6.
Lu Z, Zhang Y, Yan X, Chen Y, Tao X, Wang J, et al. Estrogen stimulates the invasion of ovarian cancer cells via activation of the PI3K/AKT pathway and regulation of its downstream targets Ecadherin and alphaactinin4. Mol Med Rep. 2014;10(5):2433-40.
Boland MJ, Nazor KL, Loring JF. Epigenetic regulation of pluripotency and differentiation. Circ Res. 2014;115(2):311-24.
Bapat SA. Epigenetic regulation of cancer stem cell gene expression. Subcell Biochem. 2013;61:419-34.
Brookes E, Shi Y. Diverse Epigenetic Mechanisms of Human Disease. Annu Rev Genet. 2014 Sep 5.
Samanidou V, Kovatsi L. Epigenetic mechanisms and therapeutic strategies. Curr Med Chem. 2011;18(12):1732.
Azad M, Kaviani S, Mortazavi Y, Norouzinia M. Evaluating the Methylation Status in Promoter of vHL, Runx3 and Ecad Genes and Expression of These Genes in Cord Blood CD34+ Stem Cells. Zanjan Uni Med Sci J. 2013;21(86):12-23
Murrell A, Hurd PJ, Wood IC. Epigenetic mechanisms in development and disease. Biochem Soc Trans. 2013;41(3):697-9.
Weishaupt H, Sigvardsson M, Attema JL. Epigenetic chromatin states uniquely define the developmental plasticity of murine hematopoietic stem cells. Blood. 2010;115(2):247-56.
Bhattacharjee S, Renganaath K, Mehrotra R, Mehrotra S. Combinatorial control of gene expression. Biomed Res Int. 2013;2013:407263.
Casimiro MC, Crosariol M, Loro E, Li Z, Pestell RG. Cyclins and cell cycle control in cancer and disease. Genes Cancer. 2012;3(11-12):649-57.
Dahmann C, Oates AC, Brand M. Boundary formation and maintenance in tissue development. Nat Rev Genet. 2011;12(1):43-55.
Rojas-Rios P, Gonzalez-Reyes A. Concise review: The plasticity of stem cell niches: a general property behind tissue homeostasis and repair. Stem Cells. 2014;32(4):852-9.
Decker J, Neuhaus C, Macdonald F, Brauch H, Maher ER. Clinical utility gene card for: von Hippel-Lindau (VHL). Eur J Hum Genet. 2014;22(4).
Avci CB, Dodurga Y, Susluer SY, Sigva ZO, Yucebas M, Caglar HO, et al. Promoter hypermethylation-mediated down-regulation of RUNX3 gene in human brain tumors. Ir J Med Sci. 2014;183(2):259-64.
Han S, Han L, Sun H, Zan X, Zhou Z, Xu K, et al. Kruppellike factor expression and correlation with FAK, MMP9 and Ecadherin expression in hepatocellular carcinoma. Mol Med Rep. 2013;8(1):81-8.