RESUMO
OBJECTIVE: Multiple myeloma (MM) is an incurable plasma cell malignancy. Several genetic and epigenetic changes affect numerous critical genes expression status in this disorder. CDKN2A gene is expressed at low level in almost all cases with MM disease. The mechanism of this gene down-regulation has remained controversial. In the present study, we targeted EZH2 by microRNA-124 (miR-124) in L-363 cells and assessed following possible impact on CDKN2A gene expression and phenotypic changes. MATERIALS AND METHODS: In this experimental study, growth inhibitory effects of miR-124 were measured by MTT assay in L-363 cell line. Likewise, cell cycle assay was measured by flowcytometery. The expression levels of EZH2 and CDKN2A were evaluated by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). RESULTS: qRT-PCR results showed induction of EZH2 gene expression after transduction of cells with lentivector expressing miR-124. The expression of CDKN2A was also upregulated as the result of EZH2 supression. Coincide with gene expression changes, cell cycle analysis by flow-cytometry indicated slightly increased G1-arrest in miRtransduced cells (P<0.05). MTT assay results also showed a significant decrease in viability and proliferation of miRtransduced cells (P<0.05). CONCLUSION: It seems that assembling of H3K27me3 mark mediated by EZH2 is one of the key mechanisms of suppressing CDKN2A gene expression in MM disease. However, this suppressive function is applied by a multi-factor mechanism. In other words, targeting EZH2, as the core functional subunit of PRC2 complex, can increase expression of the downstream suppressive genes. Consequently, by increasing expression of tumor suppressor genes, myeloma cells are stopped from aberrant expansions and they become susceptible to regulated cellular death.
RESUMO
A predominant challenge in developing curative leukemia therapy is interactions of leukemic cells with the bone marrow stromal microenvironment. We aimed to investigate the role of stromal cells, such as bone marrow mesenchymal stromal cells (BMSCs) and osteoblasts (OBs), in curcumin (CUR) and daunorubicin (DNR) induced apoptosis of acute myeloid leukemia (AML) cells. We used KG1 and U937 as leukemia cell line models and treated them with CUR and DNR. The cells were then co-cultured with BMSCs or a combination of BMSCs and OBs as feeders. After 24 hours of co-culture, BMSCs or OBs were sorted and separated from the leukemia cells and apoptosis levels were analyzed by annexin/propidium iodide (PI) staining on flow cytometry. Potentially involved molecular pathways were analyzed at gene and protein levels by Real time PCR and western blotting, respectively. The results showed AML cells cocultured with BMSCs plus OBs to be more resistant to drug induced-apoptosis compared to co-culture with BMSCs alone or without co-culture. Expression levels of OPN, CXCL-12, IL-6, STAT-3 and VCAM-1 were also significantly up-regulated in OBs and AML cells, at both mRNA and protein levels after co-culture, with concurrent enrichment of CD34+ AML cells. Our data showed, in a stromal cell niche-based model, that OBs revoke the influence of BMSCs on leukemic cells and promote enrichment of both CD34+ and CD34- leukemic stem cell (LSC) compartments in response to CUR and DNR. Up-regulation of OPN, CXCL-12, IL-6, STAT-3 and VCAM-1 in OBs and AML cells in co-culture might be part of molecular mechanisms that block CUR or CUR+DNR-induced apoptosis and promote enrichment of CD34+ and CD34- LSCs.
