The Effect of Hypoxia on molecuar and genetical aspects of MAT2A Gene in Chronic Myelogenous Leukemia cell line-K562 Co-cultured on Bone Marrow Mesenchymal

Abstract

Mesenchymal stem cells (MSCs) are one of the most prominent cells in the bone marrow (BM) milieu with a gorgeous aptitude for differentiation into a variety of cell lineages. MSCs can affect chronic myeloblastic leukemia (CML) cells under hypoxic conditions, but the way they act on the CML cells remained vague. Accordingly, we hypothesized their mechanism of action to be rather related to a characteristic feature of malignant tumors; the hypoxic microenvironment Methods: To investigate the hypothesis, we used K562 cells as simulators of an CML cancer in the in-vitro model and co-cultured with BMMSCs under hypoxic conditions. K562 cells were co-cultured with BMMSCs under the hypoxic condition induced by Cobalt Chloride (100 µM CoCl2) for 24, 48, 72, and 96 hours. Then, mRNA and protein expression of the MAT2A gene were evaluated by real-time PCR which also followed by apoptosis measurement by a flow-cytometric method based on annexin-V/PI staining. Next, the methylation status of the target genes was investigated by methylation-specific quantitative polymerase chain reaction (MS-qPCR). Results: We found that the mRNA expression of the candidate genes was strongly augmented under the hypoxic condition. Also, in experiments with the hypoxic condition, the gene expression of MAT2A was increased significantly in co-cultured cancer cells compared to K562 sole cultures (p<0.05). Moreover, we found the strong associations between the gene expression and promoter methylation levels of MAT2A gene. However, there was no relationship between the establishment of hypoxic conditions and the induction of apoptosis in K562 cells co-cultured with MSCs when compared to the apoptosis level in K562 only cultures.