Annexin A5基因沉默对Hep-2细胞凋亡的影响

Inhibition of Hep-2 cell apoptosis after annexin A5 knockdown

目的 探索annexinA5基因及其表达沉默后对人喉癌Hep-2细胞凋亡的影响.方法 培养人喉癌细胞Hep-2,经Lip2000转染特异siRNA片段沉默annexin A5基因;TRlzol法提取细胞总RNA;RT-PCR法检测annexinA5在mRNA水平的表达是否降低;Western blot法检测annexin A5的蛋白表达是否下降;经流式细胞仪AnnexinV-FITC法检测annexin A5基因沉默后的人喉癌Hep-2细胞的凋亡情况.采用SPSS 13.0软件对数据进行单因素方差分析.结果 特异siRNA片段成功转入Hep-2细胞;RT-PCR结果显示实验siRNA组、阴性siRNA组、脂质体组及空白细胞组的相对灰度值为0.70±0.03、1.18±0.05、1.17±0.06及1.23±0.07;经统计学分析表明实验组annexin A5在mRNA水平的灰度明显弱于阴性siRNA组（t=-14.77）、脂质体组（t=-13.23）及空白细胞组（t=-12.99）,P值均＜0.05;Western blot结果显示实验siRNA组、阴性siRNA组、脂质体组及空白细胞组的相对灰度值为1.21±0.03、3.88±0.06、3.87±0.02及3.95±0.08;统计学分析表明annexin A5的蛋白的相对灰度值明显弱于阴性siRNA组（t=-70.34）、脂质体组（t=-150.62）及空白细胞组（t=-56.32）,P值均＜0.05;AnnexinV-FITC法结果当annexin A5基因沉默后,人喉癌细胞Hep-2凋亡率实验siRNA组、阴性siRNA组、脂质体组及空白细胞组分别为4.43％±0.12％、13.67％±0.22％、13.66％±0.12％及13.35％±0.13％,实验组细胞凋亡率较阴性siRNA组（t=-62.50）、脂质体组（t=-14.16）及空白细胞组（t=-11.47）明显降低,P值均＜0.05.结论 在人喉癌Hep-2细胞中,annexin A5可能促进凋亡,有可能成为喉癌诊断、治疗及预后的新的生物靶标.

Objective To study the effect of annexin A5 on the apoptosis of laryngeal cancer cells.Methods Special siRNAs were used to knock annexinA5 down in Hep-2 cell,and RT-PCR and Western blot were applied to identify the efficacy of RNA interference.The flow cytometry assay was performed to detect the Hep-2 cell apoptosis.Results RT-PCR analysis showed that the relative mRNA expression of annexin A5 in siRNA group,negative control group,Lipofectamine2000 group and blank control group were 0.70 ±0.03,1.18 ±0.05,1.17 ±0.06 and 1.23 ±0.07.The relative mRNA expression of annexin A5 in siRNA group was significantly decreased than contrast groups（t =-14.77,t =-13.23,t =-12.99,P ＜0.05）.In Western blot assay,the trend of protein expression level was consistent with the mRNA expression levels of annexin A5.The relative levels of proteins in siRNA group,negative control group,Lipofectamine2000 group and blank control group were shown 1.21 ± 0.03,3.88 ± 0.06,3.87 ±0.02 and 3.95 ± 0.08.The relative protein expression of annexin A5 in siRNA group was significantly decreased than contrast groups（t =-70.34,t =-150.62,t =-56.32,P ＜0.05）.At the same time in flow cytometry the apoptotic rate of siRNA group,negative control group,Lipofectamine2000 group and blank control group were 4.43％ ±0.12％,13.67％ ±0.22％,13.66％ ±0.12％ and 13.35％ ±0.13％,the difference between the siRNA group and contrast groups was statistically significant （t =-62.50,t =-14.16,t =-11.47,P ＜ 0.05）.So after RNA interference,expression of annexin A5 decreased,and the results in the apoptosis inhibition of Hep-2 cell.Conclusion Annexin A5 promotes apoptosis of Hep-2 cells,and it may be a potential therapeutic target for the laryngeal cancer.