Correlation between methylation and expression level of P15 and P16 genes during differentiation of cord blood stem cells into erythroid lineage mediated by erythropoietin

Several influential factors such as transcription factors and intracellular signaling components are involved in differentiation of stem cells into a specific lineage. P15 and p16 proteins are among these factors. Accumulating evidences has introduced the epigenetic as a master regulator of these factors during lineage specification. The main objective of this study is to determine the correlation between the expression level and methylation pattern of P15 and P16 genes in erythroid lineage after in vitro differentiation by erythropoietin [EPO]. The purified and expanded CD34+ cord blood stem cells were differentiated into erythroid lineage in the presence of EPO. DNA was isolated from both cord blood stem cells and differentiated cells. The Real-Time PCR performed using cDNA and the isolated DNA was used in methylation Specific PCR [MSP] reaction for methylation pattern analysis in both pre and post differentiation stages. The study demonstrated that P15 and P16 genes have partial methylation after erythroid differentiation by EPO. The Expression of P15 gene was higher after differentiation and the expression of P16 gene had a slightly decreased level in post differentiation stage. Significant increase in P15 gene expression after differentiation to erythroid lineage, suggests the remarkable efficacy of this gene in erythroid function. According to upregulation of P15 gene after differentiation despite unchanged methylation status and slight down regulation of P16 gene with slight hyper-methylation of the gene it can be suggested that although the methylation can affects the expression level of P16 gene, the P15 gene is not affected by this mechanism during erythroid differentiation mediated by EPO

Ineffectiveness of methylation in rgulation of VHL, ECAD, and RUNX3 genes in erythroid cells differentiated by erythropoietin

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. 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. 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. 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

Challenges in the treatment of Iranian patients with leukemia in comparison with developed countries from the perspective of specialists

Evaluation of the factors associated with treatment process of leukemia and comparison with current related approaches in developed countries can present a good indicator to assess the weak and strong points in healthcare system of our country in leukemia treatment. The objective of this research is general and specific description of the challenges and shortcomings in Iranian healthcare system and monitoring of hematologic malignancies as well as comparison with developed countries. Our study is a descriptive-cross-sectional study. 100 hemato-oncologist , pathologists, and faculty members throughout the country were selected by random cluster sampling. Data collected using questionnaires with Cronbach's alpha coefficient of 0.76 . SPSS and Chi-square test were used for data analysis. According to the specialists, lack of advanced diagnostic facilities as well as cell and BM banks together with high treatment expenses are the main factors contributing to poor treatment processes in Iran, which are far from worldwide standards. The use of novel currently methods used in developed countries for leukemia treatment, financial and psychological support of patients under treatment , making underprivileged provinces well-equipped ,balanced specialist service distribution relative to capital city either in diagnosis or treatment are factors which makes system standardized. Moreover, integrated institutional work in relation to leukemia incidence and statistical analysis of mortality and morbidity rate can pave the way for reducing and eliminating the problems in diagnosis and treatment of leukemia patients