MicroRNA-125a regulates proliferation and apoptosis of acute myeloid leukemia through targeting NF-κB pathway.

OBJECTIVE: To elucidate the influence of microRNA-125a on the biological behaviors of acute myeloid leukemia (AML) cells. MATERIALS AND METHODS: MicroRNA-125a mimic and negative control (NC) were constructed and transfected into AML cell line HL60, respectively. Cell viability of HL60 cells transfected with microRNA-125a mimic or NC was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Regulatory effects of microRNA-125a on enzyme activities of B-cell lymphoma-2 (Bcl-2), Bcl-xl, caspase-3, and caspase-9 in HL60 cells were quantified by a spectrophotometry. Changes in apoptosis and invasion of HL60 cells overexpressing microRNA-125a were detected by flow cytometry and transwell assay, respectively. Protein levels of cell cycle genes (cyclin B, cdc-2, mdm-2), pro-apoptotic gene p53 and anti-apoptotic gene Bcl-2 in HL60 cells transfected with microRNA-125a mimic or NC were assessed by Western blot. Finally, the mRNA levels of Bax, caspase-8, nuclear factor-κB (NF-κB), and c-myc in HL60 cells with microRNA-125a overexpression were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). RESULTS: MicroRNA-125a expression remarkably increased by transfection of microRNA-125a mimic into HL60 cells, suggesting its sufficient transfection efficacy. MTT assay revealed an inhibited viability after microRNA-125a overexpression. Transfection of microRNA-125a mimic markedly enhanced enzyme activities of caspase-3 and caspase-9, but reduced activities of Bcl-2 and Bcl-xl in HL60 cells than controls (p<0.05). Moreover, microRNA-125a overexpression elevated apoptotic rate as FCM data indicated. Transwell assay demonstrated a decrease in the invasive rate of HL60 cells overexpressing microRNA-125a. Western blot analyses revealed that cell cycle genes all downregulated by transfection of microRNA-125a mimic in HL60 cells. The protein level of p53 upregulated and Bcl-2 downregulated in HL60 cells overexpressing microRNA-125a (p<0.05). Furthermore, mRNA levels of pro-apoptotic genes Bax and caspase-8 were enhanced after microRNA-125a overexpression, while mRNA levels of NF-κB and c-myc were reduced (p<0.05). CONCLUSIONS: MicroRNA-125a inhibits proliferative and invasive potentials, arrests the cell cycle in the G2/M phase of AML cells by regulating the NF-κB pathway.

Complete sequence analysis of mitochondrial DNA of aplastic anemia patients.

This study was primarily undertaken to test the hypothesis that mitochondrial DNA (mtDNA) mutations may be associated with aplastic anemia (AA). We analyzed mtDNA sequences from 15 patients with AA. The samples were obtained from bone marrow, and patients' oral epithelial cells were collected for normal tissue comparison. Total DNA was amplified by PCR after extraction, and these segments were then sent for sequencing. The results were compared with those of oral epithelial tissues as well as mtDNA sequences in the revised Cambridge Reference Sequence (rCRS) database. We detected 61 heteroplasmic mutations in 11 genes, including those encoding NADH dehydrogenase (ND)1-2 and 4-6, tRNA glutamic acid (TRNE), ribosomal RNA (RNR) 1 and 2, cytochrome c oxidase (COX1), cytochrome b (CYTB), and tRNA glycine (TRNG); mutation rates were particularly high in ND2 (34.4%) and ND4 (21.3%) in the patients' mtDNA genomes. The products of these genes are involved in oxidation in the respiratory chain, and a large number of homoplasmic mutations were found. Interestingly, these 162 polymorphisms were mostly in the D-loop DNA structure (54.3%), in which numerous mutations associated with leukemia and myelodysplastic syndromes are found. We conclude that functional impairment of the mitochondrial respiratory chain induced by mutation may be an important reason for hematopoietic failure in AA patients.