短发卡RNA抑制survivin表达并诱导CNE-2细胞凋亡

摘要

To evaluate the effect of short hairpin RNA (shRNA) silencing survivin gene on the apoptosis and proliferation of nasopharyngeal carcinoma CNE-2 cells. Methods The plasmid pSIREN-shRNA/ survivin was constructed and transfected into nasopharyngeal carcinoma CNE-2 cells. There were four groups, group A: blank control ones, group B: pSIREN-survivin/nonsense shRNA transfection, group C; pSIREN-shRNA/ survivin transfection after 24 h, group D: pSIREN-shRNA/survivin transfection after 48 h. The expression levels of survivin mRNA and protein were detected by RT-PCR and Wescern blot respectively. Apoptosis rate and cell cycle were measured by flow cytometry. Cellular proliferation was measured by MTT. Results ShRNA was transfected into CNE-2 cells effectively and transfection rate was (70. 9±4. 76)% observed with fluorescence microscopy. Survivin mRNA and protein expressions were inhibited in group C, and the inhibition rate was (41.58±0. 63)% and (68.29±0.52)%, respectively. The inhibition rate in group D was (63. 64 ± 0. 96)% and (70.83±0.48)%, respectively. The number of S phase cells decreased while that of G2/M phase cells increased through flow cytometry analysis. The apoptosis rate was (36.24+0.78)% and (50. 37±0.85)%, significantly higher than those in the negative and control groups. MTT test showed that the proliferation of CNE-2 cells was inhibited after transfection. Conclusion Short hairpin RNA silencing survivin gene can effectively inhibit expressions of survivin mRNA and protein and induce the apoptosis of CNE-2 cells, thus inhibiting their proliferation.%目的 观察以短发卡RNA(shRNA)抑制鼻咽癌细胞survivin表达对细胞凋亡与增殖的影响.方法 构建特异性survivin shRNA,转染CNE-2细胞.分组:A组为空白对照组；B组为阴性干扰组,转染pSIREN-survivin/nonsenseshRNA;C组为阳性干扰24 h组,转染pSIREN-survivin/shRNA;D组为阳性干扰48 h组,转染pSIREN-survivin/shRNA.以RT-PCR、Western-blot分别测定细胞survivin mRNA及蛋白,PI、TUNEL及MTT检测细胞周期、凋亡率、细胞增殖.结果 转染效率约(70.90±4.76)％；C组survivin mRNA和蛋白抑制率分别为(41.58±0.63)％、(68.29±0.52)％；D组抑制率分别为(63.64±0.96)％、(70.83±0.48)％.C组凋亡率为(36.24±0.78)％,D组为(50.37±0.85)％,显著高于B组和A组；S期细胞减少,G2/M期比例增高；MTT结果提示细胞增殖受到明显抑制.结论 特异性shRNA可有效干扰CNE-2内survivin的表达,诱导细胞凋亡并抑制其过度增殖.