Title of article :
Gene Expression and Promoter Methylation Status of VHL, Runx-3, E-cadherin, P15 and P16 Genes During EPO-Mediated Erythroid Differentiation of CD34+ Hematopoietic Stem Cells
Author/Authors :
Eskandari, Fatemeh Department of Hematology - School of Medical Sciences - Tarbiat Modares University, Tehran , Kalantari, Nasim Department of Hematology - School of Medical Sciences - Tarbiat Modares University, Tehran , Pourkarim, Hoda Department of Hematology - Allied Medical School - Tehran University of Medical Sciences, Tehran , Sahmani, Mehdi Department of Clinical Biochemistry - Cellular and Molecular Research Center, Qazvin , Goudarzi, Mehdi Department of Microbiology - School of Medicine - Shahid Beheshti University of Medical Science, Tehran , Mobarra, Naser Stem cell Research Center - School of Medicine - Golestan University of Medical Sciences, Gorgan , Dehghanifard, Ali Sarem Cell Research Center - Sarem Women’s Hospital, Tehran , Khamisipour, Gholamreza Department of Hematology - Bushehr University of Medical Sciences, Bushehr , Rahmani, Hanieh Department of Pharmacodynamics and Toxicology - School of Pharmacy - Mashhad University of Medical Sciences , Azad, Mehdi Department of Medical laboratory sciences - Qazvin University of Medical Sciences, Qazvin
Abstract :
Background: VHL (von Hippel-Lindau), Runx-3 (Runt-related transcription factor 3), E-cadherin (Epithelial
cadherin), P15 (INK4a, cyclin dependent kinase inhibitor), and P16 (INK4b) genes are essential in
hematopoiesis. The aim of this study was to explore the correlation between gene expression and promoter
methylation in CD34+ stem cells before and after differentiation to erythroid lineage.
Materials and Methods: CD34+ hematopoietic stem cells were separated from umbilical cord blood using
MidiMacs (positive selection) system. Expanded CD34+ stem cells were differentiated into erythroid lineage
with human recombinant erythropoietin (EPO). DNA extraction was done by QIAamp DNA Mini Kit. RNA
was extracted using RNase Mini plus Kit. MSP (Methylation specific PCR) technique was done for methylation
assay. Methylation status and expression assay was done for VHL, Runx-3, E-cadherin, P15, and P16 genes on
both CD34+ stem cells and differentiated erythroid cells.
Results: The results showed that, before differentiation, P15 had comparative methylation pattern and average
expression and it remained unchanged after differentiation (p=0.01). concerning P16, results revealed no
methylation pattern and complete expression in absence of EPO and with EPO it changed to comparative status
(p=0.01). E-cad and Runx-3 genes had relative methylation pattern and fully expression before and after
differentiation but their expression after that, was increased and decreased Respectively (p=0.04). VHL gene
had no significant methylation status before or after differentiation and its expression was complete (p=0.01).
Conclusion: The obtained results indicated that promoter methylation of P15, P16, VHL, Runx3 and E-cad was
one of the definitive expression control mechanism of these genes.
