RNA干扰抑制RAD51基因表达对放射敏感性影响的实验研究

Research on the radiosensitivity by RNAi targeting RAD51 gene

目的:构建针对DNA双链断裂修复基因RAD51的siRNA表达质粒,观察对肝癌细胞株HepG-2放射敏感性的影响。方法:采用基因重组技术,构建针对RAD51基因的干扰质粒pSilencer-1、pSilencer-2、pSilencer-3及对照质粒pSilencer-C,经脂质体转染HepG-2细胞,200μg／mL潮霉素筛选稳定表达的细胞,用Western blot测定RAD51蛋白表达量的改变,用细胞克隆形成实验测定细胞放射敏感性。结果:筛选出稳定转染各组质粒的细胞;转染pSilencer -1、pSilencer-2和pSilencer-3质粒细胞克隆的RAD51蛋白表达量分别为转染pSilencer-C细胞克隆的0．60±0．29、0．36±0．16和0．15±0．09,t值分别为4．101、6．561和8．714,P值分别为0．049、0．003和0．001。pSilencer-C组、pSilencer-2组和pSilencer-3组三组剂量存活曲线的D0值分别为1．55、1．46和1．44,Dq值分别为3．31、3．16和2．82,SF2分别为0．82、0．73和0．67 Gy;与pSilencer-C组相比,pSilencer-2组和pSilencer-3组在2 Gy剂量时的放射增敏比SERSF2分别为1．12和1．23。结论:RNAi抑制RAD51基因的表达,能提高HepG-2细胞的放射敏感性,提示RAD51基因可作为放射增敏治疗的一个潜在靶点。

OBJECTIVE： To construct recombinant plasmids generating short interfering RNA targeting DSB repair gene RAD51 and investigate the effects on the radiosensitivity of human hepatocellular carcinoma cells. METHODS： By the molecular cloning technique pSilencer-1, pSilencer-2, pSilencer-3 and negative control plasmid pSilencer-C were constructed. HepG-2 cells were transfected with recombinant plasmids by Lipofectamine 2000 following selection by hygromycin 200 μg/mL. The level of RAD51 protein was assessed by Western blot. The cellular radiosensitivity was measured by clonogenic survival assays. RESULTS： The cell clones that stably expressed siRNAs targeting RAD51 were selected. The levels of RAD51 protein of pSilencer-1, pSilencer-2 and pSilencer-3 reduced to 0. 60±0. 29 （t=4. 101 ,P=0.049）, 0. 36±0.16 （t= 6. 561,P=0. 003） and 0.15±0.09 （t=8. 714,P=0. 001） respectively, compared to pSilencer-C. The Do values of pSilencer-C,pSilencer-2 and pSilencer-3 groups were 1.55,1.46 and 1.44 respectively. The Dq values were 3.31,3.16 and 2.82 respectively. The values of SF2 were 0.82 Gy,0.73 Gy and 0.67 Gy respectively. SERSFZ（s） of pSilencer-2 and pSilencer-3 groups were 1.12 and 1.22 respectively, compared to pSilencer-C group. CONCLUSION：The reduction of RAD51 by RNAi can enhance the radiosensitivity of HepG-2 cancer cells, which suggests that RAD51 can be a candidate target for tumor gene therapy.