CAJ | 학술논문

目的研究以microRNA-17(miR-17)为靶点的反义核酸对人类白血病K562细胞的生长抑制作用.方法人工合成miR-17的反义核酸,硫代修饰,用Lipofectamine 2000转入K562细胞.四甲基偶氮唑蓝(MTT)比色法检测转染反义核酸后K562细胞的增殖活性,流式细胞术(FCM)检测细胞的凋亡水平,实时荧光定量聚合酶链反应(PCR)检测反义核酸作用后K562细胞内miR-17的相对表达水平.结果 MTT结果显示,转染反义核酸后的24、48、72 h,K562细胞的增殖活性分别为0.872±0.001、0.710±0.002、0.551±0.001,与随机核酸序列组(随机对照组)(增殖活性分别为1.001±0.002、1.009±0.003、1.211±0.003；t值分别为182.58、269.77、660.40)、空白对照组(增殖活性分别为1.113±0.001、1.114±0.001、1.101±0.001；t值分别为537.98、571.20、1230.51)相比较差异有统计学意义(均P＜0.05)；FCM检测结果表明,在转染反义核酸48 h后细胞的凋亡率为(20.14±0.01)％,与随机对照组的(3.54±0.02)％及空白对照组的(1.98±0.01)％比较差异有统计学意义(t分别为2347.6、2568.2,均P＜ 0.01)；实时荧光定量PCR结果证实,在反义核酸作用后,K562细胞内miR-17的相对表达水平(0.07)明显下降,与随机组(1.00)、空白组(1.01)相比较差异有统计学意义(t值分别为148.63、147.04,均P＜0.05).结论以miR-17为靶点的反义核酸可抑制K562细胞的增殖活性,并显著促进细胞的凋亡.
목적 연구이microRNA-17(miR-17)위파점적반의핵산대인류백혈병K562세포적생장억제작용.방법 인공합성miR-17적반의핵산,류대수식,용Lipofectamine 2000전입K562세포.사갑기우담서람(MTT)비색법검측전염반의핵산후K562세포적증식활성,류식세포술(FCM)검측세포적조망수평,실시형광정량취합매련반응(PCR)검측반의핵산작용후K562세포내miR-17적상대표체수평.결과 MTT결과현시,전염반의핵산후적24、48、72 h,K562세포적증식활성분별위0.872±0.001、0.710±0.002、0.551±0.001,여수궤핵산서렬조(수궤대조조)(증식활성분별위1.001±0.002、1.009±0.003、1.211±0.003；t치분별위182.58、269.77、660.40)、공백대조조(증식활성분별위1.113±0.001、1.114±0.001、1.101±0.001；t치분별위537.98、571.20、1230.51)상비교차이유통계학의의(균P＜0.05)；FCM검측결과표명,재전염반의핵산48 h후세포적조망솔위(20.14±0.01)％,여수궤대조조적(3.54±0.02)％급공백대조조적(1.98±0.01)％비교차이유통계학의의(t분별위2347.6、2568.2,균P＜ 0.01)；실시형광정량PCR결과증실,재반의핵산작용후,K562세포내miR-17적상대표체수평(0.07)명현하강,여수궤조(1.00)、공백조(1.01)상비교차이유통계학의의(t치분별위148.63、147.04,균P＜0.05).결론 이miR-17위파점적반의핵산가억제K562세포적증식활성,병현저촉진세포적조망.
Objective To explore the inhibitory effect of anti-miRNA-17 oligonucleotide on leukemic K562 cells.Methods K562 cells were transfected with anti-miRNA-17 oligonucleotide,cell viability was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-zoliunbromide (MTT) assay.Apoptosis was detected by flow cytometry,expression of miRNA-17 in K562 cells was measured by real-time PCR.Results MTT results showed transfection of antisense nucleic acid significantly decreased cell proliferation activity,after 24,48,72 h they were respectively 0.8719±0.001,0.7102±0.002,0.5507±0.001,the difference being statistically significant (P ＜ 0.05) when compared to the random control (t =182.575,269.77,660.4) or control group (t =537.98,571.20,1230.51).FCM test results showed that after 48 h of transfecting antisense nucleic acid apoptosis rate was (20.14 ± 0.01) ％,and was statistically significant compared to the randomized control or blank control group (t =2347.6,2568.2,P ＜ 0.01).Fluorescence real-time quantitative PCR confirmed antisense nucleic acid significantly decreased the relative expression level of miR-17 in K562 cells (0.07).The difference was statistically significant compared to the random group or blank group (1,1.01) (t =148.63,147.04,P ＜ 0.05).Conclusion Targeted inhibition of miR-17 with oligonucleotide can suppress K562 cell growth and induce apoptosis.