靶向生存素基因的RNAi对肺腺癌A549细胞放射敏感性的影响

目的 构建靶向生存素(survivin)基因的RNA干扰(RNAi)载体,观察对肺腺癌A549细胞放射敏感性的影响,并探讨其机制.方法 根据survivin的cDNA序列设计干扰序列,构建干扰survivin的重组干扰质粒pGenesil2-survivin.酶切、测序鉴定正确后,经脂质体介导转染肺腺癌A549细胞;应用反转录.聚合酶链式反应(RT-PCR)和Western blot法检测survivin的表达;流式细胞术检测细胞凋亡的变化;克隆形成实验检测细胞的放射敏感性.结果 酶切和测序结果显示,载体构建正确;将质粒pGenesil2-survivin转染肺腺癌A549细胞48 h,survivin蛋白水平及mRNA水平在正常组与pGenesil2组中无明显变化,5 Gy照射组增强,而转染pGenesil2-survivin后明显抑制;pGenesil2-survivin与5 Gy X射线照射都能诱导细胞凋亡增加(t1=10.63,P<0.001;t2=3.75,P<0.05),两者共同作用,凋亡增加更明显(t=4.83,P<0.05);克隆形成实验显示,pGenesil2与正常组D0、Dq无明显变化,而pGenesil2.survivin则明显降低,说明其可以提高A549细胞的放射敏感性.结论 靶向生存素基因的RNAi能够明显抑制survivin mRNA与蛋白的表达,促进凋亡,增强A549细胞的放射敏感性.

Effects of RNAi targeting survivin gene on A549 cell radiosensitivity

Objective To construct the RNAi vector targeting survivin gene, in order to observe its effect on lung adenoearcinoma A549 cell radiosensitivity, and explore the possible mechanism. Methods One pair of RNA interference sequence targeting survivin gene was designed according to the eDNA sequence, the recombinant RNAi plasmid pGenesil2-survivin was constructed. After certified by enzyme digestion and sequencing, the pGenesil2-survivin plasmid was transfected into A549 cells. Survivin mRNA and protein was measured by RT-PCR and Western blot, respectively. The apoptosis was analyzed with flow cytometry. The radiosensitivity was measured by clone formation assay. Results The pGenesil2-survivin vector was successfully established. After the pGenesil2-survivin was transfeeted into A549 cells for 48 h, surviving protein and mRNA in pGenesil2 group were not obviously changed compared with that in normal group. They were increased after 5 Gy X-ray irradiation, but obviously inhibited in pGenesil2-survivin group. The cell apoptosis in pGenesil2-survivin and 5 Gy X-ray groups was obviously increased (t1 = 10.63, P < 0.001 ; t2 = 3.75 , P < 0.05), the effect was more manifest in combined group(t = 4.83, P < 0.05). D0 and Dq in normal and pC, enesil2 group were not obviously different, but those in pGenesil2-survivin group were lower. Conclusions RNAi targeting survivin gene could inhibit survivin mRNA and protein expression, enhance the A549 cell apoptosis rate and cell radiosensitivity.