Hsa-miR-204反义核酸提高MOLT-4细胞对苦参碱的敏感性研究

AMO-miR-204 antisense oligonucleotides enhance the sensitivity of MOLT-4 cells to matrine

目的研究miR-204的反义核酸(anti-miR204 oligonucleotides,AMO-miR-204)联合苦参碱观察其对人急性淋巴细胞白血病细胞株(MOLT-4)生长与凋亡的影响及机制。方法将一定浓度的人工合成的AMO-miR-204经脂质体包裹转染MOLT-4细胞,并联合不同浓度的苦参碱作用于MOLT-4细胞48 h后,采用MTT法检测单用AMO-miR-204、单用苦参碱及AMO-miR-204联合苦参碱对MOLT-4细胞增殖抑制作用,流式细胞术检测细胞早期凋亡率;实时荧光定量RT-PCR检测细胞Bcl-2mRNA的表达水平;克隆形成抑制实验检测克隆形成能力。结果单用苦参碱的IC50为0.75 mg·L-1;苦参碱与阴性对照联合使用,IC50为0.29 mg·L-1,表现为相加作用;苦参碱与AMO-miR-204联合使用,IC50为0.07 mg·L-1,增敏倍数为10.7,表现为协同作用。流式细胞术结果显示:联合组比单用组早期凋亡率明显增高,凋亡率达25.4%。单用AMO-miR-204、苦参碱及两者联均能下调Bcl-2mRNA基因的表达水平,对MOLT-4细胞克隆形成逐渐减小,其中两者联合的克隆数为(28±3.0)。结论 AMO-miR-204可提高MOLT-4细胞对苦参碱的敏感性,其机制可能为通过下调Bcl-2 mRNA的表达水平,促进MOLT-4细胞早期凋亡,并抑制其克隆形成有关。

Objective To study the effect of antisense oligonucleotides targeted on has-miR-204（anti-miR204 oligonucleotides,AMO- miR-204） for enhancing the matrine sensitivity of acute lymphoblastic leukemia cells and its possible acting mechanism. Methods Do- sing and ehemosynthetie antisense oligonucleotides targeted on has-miR-204 was transfeeted to the cells using Lipofeetamine TM 2000 and combined with different concentrations of matrine. The inhibitor7 effects of matrine and AMO-miR-204,used singly or in combina- tion,on cell proliferation were detected by Mq^f and clone formation assay. The expression of Bcl-2 mRNA was analyzed by real time fluorescent quantitative reverse transcription polymerase chain reaction. The percentage of apoptosis cells was determined by flow eytom- ett3,. Results Used in combination with AMO-miR-204,the iCsoof matrine could be lowered from 0.75 nag ~ L ＊to 0.07 nag ~ L ＊, and the sensitivity of cells to procyanidin increased to 10.7. The amount of apoptotic cells increased significantly. The early apoptotic rate was 25.4%. Transfection with AMO-miR-204 alone or combind with matrine could down-regulate the expression of Bcl-2 mRNA in cells. The colon formation ability was decreased. Conclusions Combined use of AMO-miR-204 could increase the sensitivity of MOLT- 4 cells to matrine. The mechanism may be to promote MOLT-4 cells early apoptosis and down-regulate the expression of Bcl-2 mRNA, which provides a novel potential approach for treatment of acute lymphoblastie leukemia.