Department of Central Laboratory, The Affiliated People's Hospital, Jiangsu University, Zhenjiang, China.
Acta Biochim Biophys Sin (Shanghai). 2012 Mar;44(3):269-77. doi: 10.1093/abbs/gmr128. Epub 2012 Jan 26.
The aim of this study was to investigate whether miR-181a could modulate the sensitivity of the leukemia drug-resistant cell line K562/A02 to the chemotherapeutic agent daunorubicin (DNR), and explore the mechanism of miR-181a on the DNR sensitivity of K562/A02 cells. MicroRNA microarray and stem-loop reverse transcription-polymerase chain reaction were used to detect the expression of miR-181a. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay was performed to quantify the effect of miR-181a on K562 cells growth and viability. Apoptotic cells were quantitatively detected using Annexin V/FITC and PI apoptosis detection kit. BCL-2 protein expression was measured by western blot. Luciferase reporter vector with the putative BCL-2 3' untranslated region was constructed to explore whether BCL-2 was a direct target gene of miR-181a. BCL-2 siRNA was transfected into the cell to explore the relationship between BCL-2 and DNR resistance. The miR-181a expression level was lower in the K562/A02 cells than in the K562 cells (P< 0.05). K562 cells that were transfected with miR-181a inhibitor had a significantly higher survival than K562 cells, and K562/A02 cells that were transfected with the miR-181a mimic had a significantly lower survival than K562/A02 cells (P< 0.05). miR-181a could enhance DNR-induced apoptosis in K562/A02 cells. BCL-2 siRNA transfected K562/A02 cells had decreased survival compared with the K562/A02 control group. In conclusion, miR-181a could play a role in the development of DNR resistance in K562/A02 cells and the over-expression of miR-181a could sensitize K562/A02 cells to DNR by targeting BCL-2.
本研究旨在探讨 miR-181a 是否能够调节白血病耐药细胞系 K562/A02 对化疗药物柔红霉素（DNR）的敏感性，并探讨 miR-181a 对 K562/A02 细胞 DNR 敏感性的作用机制。采用 miRNA 微阵列和茎环反转录聚合酶链反应检测 miR-181a 的表达。3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-2H-四唑溴盐（MTT）比色法检测 miR-181a 对 K562 细胞生长和活力的影响。采用 Annexin V/FITC 和 PI 凋亡检测试剂盒定量检测凋亡细胞。采用 Western blot 法检测 BCL-2 蛋白表达。构建包含 BCL-2 3'非翻译区的荧光素酶报告载体，探讨 BCL-2 是否为 miR-181a 的直接靶基因。转染 BCL-2 siRNA 到细胞中，探讨 BCL-2 与 DNR 耐药的关系。与 K562 细胞相比，K562/A02 细胞中 miR-181a 的表达水平较低（P<0.05）。转染 miR-181a 抑制剂的 K562 细胞的存活率明显高于 K562 细胞，转染 miR-181a 模拟物的 K562/A02 细胞的存活率明显低于 K562/A02 细胞（P<0.05）。miR-181a 可增强 DNR 诱导的 K562/A02 细胞凋亡。与 K562/A02 对照组相比，转染 BCL-2 siRNA 的 K562/A02 细胞的存活率降低。综上所述，miR-181a 可能在 K562/A02 细胞中 DNR 耐药的发展中发挥作用，过表达 miR-181a 可能通过靶向 BCL-2 使 K562/A02 细胞对 DNR 敏感。
