CAJ | 학술논문

目的探讨以微小RNA-21(microRNA-21,miRNA-21)为靶标的反义核酸(AMO-miR-21)提高白血病K562细胞对三氧化二砷(As_2O_3)的敏感性及可能的作用机制.方法利用LipofectamineTM 2000将化学合成的反义核酸转染K562细胞.MTT法检测单独使用AMO-miR-21、As_2O_3以及两者联合使用对细胞增殖的抑制作用,并计算抑制率和IC_(50);PI单染检测细胞周期及凋亡;实时定量PCR检测miRNA-21表达水平的改变;免疫荧光法检测miRNA-21候选靶基因PDCD_4蛋白表达的改变.结果 AMO-miR-21与As_2O_3联合使用后,IC_(50)从2.10 μmol/L降低到1.23 μmol/L,敏感性提高到单用As_2O_3的1.78倍;AMO-miR-21联合As_2O_3组细胞凋亡明显增加(P<0.05);单独转染AMO-miR-21可显著抑制K562细胞中miRNA-21的表达水平(P<0.01),同时抑癌基因PDCD_4蛋白表达明显增加(P<0.05).结论 AMO-miR-21与As_2O_3联合使用可提高K562细胞对As_2O_3的敏感性,为白血病的治疗提供了新的方法.AMO-miR-21通过靶向抑制miRNA-21,进而上调抑癌基因PDCD_4的表达而发挥抗肿瘤作用.
목적 탐토이미소RNA-21(microRNA-21,miRNA-21)위파표적반의핵산(AMO-miR-21)제고백혈병K562세포대삼양화이신(As_2O_3)적민감성급가능적작용궤제.방법 이용LipofectamineTM 2000장화학합성적반의핵산전염K562세포.MTT법검측단독사용AMO-miR-21、As_2O_3이급량자연합사용대세포증식적억제작용,병계산억제솔화IC_(50);PI단염검측세포주기급조망;실시정량PCR검측miRNA-21표체수평적개변;면역형광법검측miRNA-21후선파기인PDCD_4단백표체적개변.결과 AMO-miR-21여As_2O_3연합사용후,IC_(50)종2.10 μmol/L강저도1.23 μmol/L,민감성제고도단용As_2O_3적1.78배;AMO-miR-21연합As_2O_3조세포조망명현증가(P<0.05);단독전염AMO-miR-21가현저억제K562세포중miRNA-21적표체수평(P<0.01),동시억암기인PDCD_4단백표체명현증가(P<0.05).결론 AMO-miR-21여As_2O_3연합사용가제고K562세포대As_2O_3적민감성,위백혈병적치료제공료신적방법.AMO-miR-21통과파향억제miRNA-21,진이상조억암기인PDCD_4적표체이발휘항종류작용.
Objective To study the effect of antisense oligonucleotide targeted on miRNA-21 (AMO-miR-21) for enhancing the arsenic trioxide (As_2O_3) sensitivity of leukemic K562 cells and its possible acting mechanism. Methods Chemosynthetic AMO-miR-21 was transfected to K562 cells using Lipofectamine TM 2000. The inhibitory effects of As_2O_3 and AMO-miR-21,used singly or in combining,on cell proliferation were detected by MTT,their inhibition rate and IC_(50) were calculated. Cell cycle and apoptosis were assessed with PI stain;expression of miRNA-21 in cells was detected quantitatively by real-time PCR,and the potential target gene PDCD_4 protein expression was detected by immuno-fluoremetry. Results Used in combining with AMO-miR-21,the IC_(50) of As_2O_3 could be lowered from 2.1 μmol/L to 1.23 μmol/L,and the sensitivity of cells to As_2O_3 increased to 1.78-fold;with the amount of apoptotic cells increased significantly. Transfection with AMO-miR-21 alone could down-regulate the expression of miRNA-21 in cells (P<0.01),and up-regulate PDCD_4 protein expression level significantly. Conclusions Combined use of AMO-miR-21 and As_2O_3 could increase the sensitivity of K562 cells to As_2O_3,which provides a novel potential approach for treatment of leukemia. AMO-miR-21 realizes it anti-tumor action by way of targeted inhibition on miRNA-21,and further up-regulates the expression of anti-tumor gene PDCD_4.