预照射联合siRNA抑制小鼠肺癌Survivin基因表达的研究

Research on the expression of Survivin gene suppressed by pre-irradiation and intratumor injection siRNA in lung cancer of mouse

背景与目的:肿瘤放化疗的效果与肿瘤细胞凋亡成正相关。Survivin是凋亡抑制基因,在肺癌中过度表达,降低其表达可增加肺癌细胞凋亡。RNA干扰可以特异、高效地封闭该基因的表达。肿瘤组织接受一定剂量的照射后,也可以提高基因转导率。本研究旨在探讨预照射联合瘤内注射si RNA对小鼠肺癌Survivin基因表达的影响。方法:将皮下移植瘤小鼠随机分为4组:未处理组(A组)、单纯si RNA瘤内注射组(B组)、单纯放射组(C组)和4 Gy预照射+si RNA瘤内注射组(D组)。小鼠经上述不同处理2 d后用颈椎脱臼法处死,剥离皮下移植瘤,采用实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)和蛋白[质]印迹法(Western blot)检测Survivin基因在m RNA和蛋白水平的表达情况;采用流式细胞术检测细胞周期及细胞凋亡。结果:B组、C组、D组Survivin基因在m RNA和蛋白水平的表达均较A组减少,且D组与A组Survivin基因在m RNA和蛋白水平的差异有统计学意义(P=0.036);D组处于S期的细胞比例为(2.70±0.34)%,较B组[(8.93±0.75)%]和C组[(6.71±0.51)%]明显减少,且与A组S期细胞的比例相比,差异有统计学意义(P=0.034);D组肿瘤组织的细胞凋亡率为(25.6±0.65)%,较其他3组肿瘤组织的细胞凋亡率明显增多,差异有统计学意义(F=78.82,P<0.05)。结论:预照射可增强si RNA的转导率,使肺癌组织中Survivin基因表达水平降低,促进细胞凋亡,进而增加放疗敏感性。

Background and purpose： The effects of tumor after radiotherapy and chemotherapy with tumor cell apoptosis have positive correlation. Survivin is an apoptosis suppressor gene, which is overexpressed in lung cancer. The reduction of its expression can increase lung cancer cell apoptosis. RNA interference can speciifcally and effectively blocked the expression of this gene. After accepting a dose of radiation, tumor tissues can raise the rate of gene transduction. The purpose of this study was to investigate the effects of the expression of Survivin gene suppressed by intratumor injection siRNA and radiation in lung cancer. Methods：Mouse subcutaneous transplanted with tumors were randomly divided into four groups：untreated group （group A）, group with intratumor injection siRNA （group B）, group with radiation （group C）, group 4 Gy pre-irradiation combination with intratumor injection siRNA （group D）. Two days later, mouse with different treatments were executed by cervical dislocation and stripped the subcutaneous tumors. mRNA and protein levels of Survivin gene were detected by quantitative real-time PCR （qRT-PCR） and Western blot; Cell cycle and cell apoptosis were assayed by lfow cytometry （FCM）. Results：The expression of Survivin gene in mRNA and protein levels in group B, C, and D were signiifcantly reduced compared with group A. The differences of Survivin expression in mRNA level between group A and group D were statistically signiifcant. Compared with the other 3 groups, the expression of Survivin gene in group D was signiifcantly reduced, the differences were statistically signiifcant （P=0.036）;The proportion of cells in S-phase of group D was （2.70±0.34）%, compared with group B [（8.93±0.75）%] and group C [（6.71±0.51）%], that was significantly reduced. The differences of the proportion of S-phase cells in group A and group D were statistically signiifcant （P=0.034）;The rate of cell apoptosis in group D was （25.67 ± 0.65）%, which was signiifcantly increased compared with the rate of cell apoptosis in the other 3 groups, the differences were statistically signiifcant （F=78.82, P〈0.05）. Conclusion：Pre-irradiation can enhance the transduction rate of siRNA, reduce the expression of Survivin gene in lung cancer, promote cell apoptosis, and increase the sensitivity of the radiotherapy.