ABSTRACT
BACKGROUND@#Acute myeloid leukemia (AML) is a malignant hematological disease, originating from hematopoiesis stem cell differentiation obstruction and clonal proliferation. New reagents or biologicals for the treatment of AML are urgently needed, and exosomes have been identified as candidate biomarkers for disease diagnosis and prognosis. This study aimed to investigate the effects of exosomes from bone marrow mesenchymal stem cells (BMSCs) on AML cells as well as the underlying microRNA (miRNA)-mediated mechanisms.@*METHODS@#Exosomes were isolated using a precipitation method, followed by validation using marker protein expression and nanoparticle tracking analysis. Differentially expressed miRNAs were identified by deep RNA sequencing and confirmed by quantitative real-time polymerase chain reaction (qPCR). Cell proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt method, and cell cycle progression and apoptosis were detected by flow cytometry. Functional gene expression was analyzed by qPCR and Western blotting (WB). Significant differences were determined using Student's t test or analysis of variance.@*RESULTS@#BMSCs-derived exosomes effectively suppressed cell proliferation (both P < 0.0001 at 10 and 20 μg/mL) and cell cycle progression (P < 0.01 at G0-G1 stage), and also significantly enhanced cell apoptosis (P < 0.001) in KG-1a cells. There were 1167 differentially expressed miRNAs obtained from BMSCs-derived exosomes compared with KG-1a cell-derived exosomes (P < 0.05). Knockdown of hsa-miR-124-5p in BMSCs abrogated the effects of BMSCs-derived exosomes in regulating KG-1a such as the change in cell proliferation (both P < 0.0001 vs. normal KG-1a cell [NC] at 48 and 72 h). KG-1a cells treated with BMSCs-derived exosomes suppressed expression of structural maintenance of chromosomes 4 (P < 0.001 vs. NC by qPCR and P < 0.0001 vs. NC by WB), which is associated with the progression of various cancers. This BMSCs-derived exosomes effect was significantly reversed with knockdown of hsa-miR-124-5p (P < 0.0001 vs. NC by WB).@*CONCLUSIONS@#BMSCs-derived exosomes suppress cell proliferation and cycle progression and promote cell apoptosis in KG-1a cells, likely acting through hsa-miR-124-5p. Our study establishes a basis for a BMSCs-derived exosomes-based AML treatment.
Subject(s)
Humans , Apoptosis/genetics , Cell Proliferation/genetics , Exosomes/genetics , Leukemia, Myeloid, Acute/genetics , Mesenchymal Stem Cells , MicroRNAs/geneticsABSTRACT
This study was aimed to investigate the effects of methylation of runx3 gene promoter on pathogenesis of acute leukemia (AL) and its clinical significance. The methylation of runx3 gene promoter in cells of bone marrow or peripheral blood from 40 cases of AL and 10 healthy persons as well as in CHRF, U937 and K562 cell lines were assuaged by methylation specific polymerase chain reaction (MS-PCR), the expression of runx3 gene were detected with reverse transcription polymerase chain reaction (RT-PCR). The results indicated that no methylation was detected in all of cell lines and healthy persons while expression of runx3 gene could be deteted, methylation of runx3 gene promoter was found in 35% (14/40) AL patients and its percentage was significant higher than that healthy persons (0%), the difference in methylation for runx3 between two kinds of samples was statistically significant (p<0.05), while methylation rate in AML was 30.43% (7/23), ALL was 41.18% (7/17), there was no significant difference between them (p>0.25). All of methylated samples had no expression of runx3 gene. Patients without methylation of runx3 gene had a lower percentage of blasts in bone marrow and a higher complete remission rate of first chemotherapy than those with methylation of runx3 gene. It is concluded that methylation of runx3 gene promoter probably plays a role in the pathogenesis of AL and may have clinical significance in predicting prognosis of AL.