靶向存活素基因的RNAi联合X线照射对肺腺癌A549细胞凋亡的影响

Effect of RNAi targeting survivin gene combined with X-rays radiation on apoptosis of lung adenocarcinoma A549 cells

目的:构建靶向存活素(survivin)基因的RNA干扰(RNAi)载体,观察其联合X射线照射对肺腺癌A549细胞凋亡的影响。方法:根据GenBank中survivin的cDNA序列设计干扰序列,构建重组干扰质粒pGenesil2-survivin。酶切、测序鉴定正确后,干扰质粒经脂质体介导转染肺腺癌A549细胞;实验设正常组、空载体组、pGenesil2-survivin组、5 Gy照射组和pGenesil2-survivin+5 Gy照射组。流式细胞术与TUNEL检测细胞凋亡的变化,Western blotting检测survivin及caspase-3蛋白表达。结果:KpnⅠ和EcoRⅠ双酶切,得到大小为389 bp和4 206 bp的两个片段,与预期结果一致;测序结果与寡核苷酸链经DNAssist2.0软件进行比对,二者完全一致,说明载体构建正确;质粒pGenesil2-survivin转染肺腺癌A549细胞48 h,pGenesil2-survivin与5 Gy X射线照射都能诱导细胞凋亡百分率增加(P<0.05),二者共同作用凋亡百分率增加更明显;Westernblotting结果显示,pGenesil2-survivin组中survivin蛋白与-βactin蛋白灰度比值较正常组明显降低(P<0.01);而caspase-3蛋白与-βactin蛋白灰度比值较正常组明显增强,且pGenesil2-survivin+5 Gy照射组增加更明显(P<0.01)。结论:靶向存活素基因的RNAi能够明显抑制survivin蛋白表达,增强caspase-3蛋白表达,促进凋亡;联合5 Gy X射线照射,增强促凋亡作用。

Objective To construct the vector of RNA interference （RNAi） targeting survivin gene and observe its effect combined with X-rays radiation on lung adenocarcinoma A549 cell apoptosis. Methods One pair of RNAi sequence targeting survivin gene were designed according to its cDNA sequence reported in GenBank, the recombinant RNAi plasmid pGenesil2-survivin was constructed. After identified by enzyme digestion and sequencing, the pGenesil2-survivin plasmid was trasfeced into A549 cells. In the experiment, normal group, pGenesil2 group, pGenesil2-survivin group, 5 Gy irradiation group and pGenesil2-survivin ＋ 5 Gy irradiation group were set up. The apoptosis of A549 cells was measured by flow cytometry with PI/Annexin V and TUNEL, the survivin and caspase 3 expressions were measured by Western blotting. Results Two fragments about 389 bp and 4 206 bp were gotten by Kpn Ⅰ and EcoR Ⅰ enzyme digestion, they are the same to expected result, the sequencing result was compared to oligonucleotide chain with DNAssist 2. 0, they were equal, these indicated the identification of pGenesil2-survivin vector was right; pGenesil2-survivin was transfected into A549 cells for 48 h, the apoptotic percentage in pGenesil2-survivin and 5 Gy X-rays groups increased obviously （P〈 0.05）, when the both were combined, the effect was more obvious; the Western blotting results appeared that the survivin gray scale/β-actin gray scale in pGenesil2-survivin group was lower than that in normal group （P〈 0. 01）, and the caspase-3 gray scale/β-actin gray scale was higher than that in normal group, and that ratio in pGenesil2-survivin＋ 5 Gy irradiation group was more high （P〈 0.01）. Conclusion RNAi targeting surviving gene could inhibit survivin protein expression, but enhance caspase-3 protein expression, and promote apoptosis. When it is combined with 5 Gy X-rays irradiation, the promotion of apoptosis is enhanced.