【摘要】 目的研究miR-21在鼻咽癌组织及细胞中的表达,并分析miR-21反义核苷酸(antisense oligonucleotide,ASO)对鼻咽癌细胞增殖和凋亡的影响。方法用荧光定量RT-PCR方法检测miR-21在鼻咽癌组织及细胞株中的表达;利用脂质体Lipofectamine TM2000将miR-21 ASO瞬时转染至鼻咽癌细胞,通过荧光定量RT-PCR检测miR-21的沉默效果。利用四甲基偶氮唑盐比色法(MTT法)、平板克隆形成实验检测miR-21对细胞增殖的影响;流式细胞术检测miR-21对细胞周期的影响;流式细胞仪检测细胞凋亡-Annexin V-FITC/PI双染色法检测miR-21对鼻咽癌细胞凋亡的影响;蛋白质印迹法(Western blot)检测caspase-3蛋白的表达;caspase-3活性检测试剂盒检测沉默miR-21后caspase-3的活性变化。结果 miR-21在鼻咽癌组织及鼻咽癌细胞中显著高表达;MTT法分析显示,细胞接种96 h后,5-8F/miR-21 ASO组的生长速度较对照组显著下降(P<0.05),细胞克隆形成率较对照组显著降低,差异具有统计学意义(P<0.05);细胞周期结果表明沉默miR-21表达的细胞增殖受抑,主要通过诱导G0/G1期阻滞,减少S期细胞的比例;流式细胞仪检测结果显示转染miR-21 ASO组较对照组细胞凋亡率明显增加,并且caspase-3的活性明显增强。结论 miR-21在鼻咽癌组织及细胞中表达上调,靶向miR-21可有效抑制鼻咽癌细胞增殖、促进细胞凋亡。miR-21的表达可能影响鼻咽癌细胞的生长。更多还原

【Abstract】 Objective To investigate the expression of miR-21 in nasopharyngeal carcinoma( NPC) and to explore the regulative effect of miR-21 antisense oligonucleotide( ASO) on the proliferation and apoptosis of NPC cells. Methods Expression of miR-21 in NPC tissues and NPC cell lines were detected by real-time RT-PCR. After transient transfection of miR-21 ASO to NPC cells with Lipofectamine TM2000,the effect of miR-21 silence on cell proliferation and cell cycle was detected by MTT assay,plate colony formation assay and flow cytometry. The cell apoptosis following miR-21 silence was detected by flow cytometry. Western blot was performed to detect the protein level of cleaved-caspase-3 and caspase-3activity was determined by caspase-3 activity assay kit. Results Expression of miR-21 was significantly increased in NPC tissues and cell lines. MTT assay showed that miR-21 downregulation significantly inhibited the growth of 5-8 F / miR-21 ASO cells. Colony formation rate of 5-8 F / miR-21 ASO cells was lower than that of the control. Cell cycle analysis showed that most 5-8 F / miR-21 ASO cells were arrested in G0 / G1 phase with a very low ratio of cells in S phase. Flow cytometry indicated that the apoptotic index in miR-21 ASO group was significantly higher than that in the control group. In addition,caspase-3 activity was obviously increased in 5-8 F / miR-21 ASO cells. Conclusions miR-21 is overexpressed in human NPC. Reducing expression of miR-21 can effectively inhibit the growth of NPC cells and promote their apoptosis. MiR-21 may become a new target for the regulation of gene expression in NPC.更多还原