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目的探讨RNA干扰沉默survivin基因对食管癌Eca-109细胞放射敏感性的影响。方法构建靶向survivin基因特定序列的小干扰RNA(small interfering,siRNA)真核表达干扰质粒pRNAT-siRNA-survivin转染食管癌Eca-109细胞为干扰组,构建阴性对照质粒pRNAT-siRNA-control转染Eca-109细胞为阴性对照组,未转染的Eca-109细胞为空白对照组,3组采用Western blot检测survivin蛋白表达水平。取对数生长期干扰组Eca-109/si-survivin细胞和空白对照组Eca-109细胞,在直线加速器下分别给予6Gy的X线照射,分别为干扰+X线照射组和单纯X线照射组(X线照射组),采用流式细胞术检测各组细胞凋亡指数,采用MTT法分析细胞存活分数。结果干扰组细胞survivin蛋白表达水平(18.75±3.12)较阴性对照组(44.17±3.15)和空白对照组(46.20±2.62)明显下调(P<0.05);干扰+X线照射组细胞凋亡指数[(29.56±0.74)%]明显高于空白对照组[(4.35±0.15)%]、阴性对照组[(4.32±0.32)%]、干扰组[(9.24±0.35)%]和X线照射组[(22.17±0.75)%],差异均有统计学意义(P<0.05),干扰组和X线照射组明显高于空白对照组和阴性对照组(P<0.05);干扰+X线照射组细胞存活分数[(19.54±1.12)%]明显低于空白对照组[(95.35±1.40)%]、阴性对照组[(93.45±1.38)%]、干扰组[(70.96±0.85)%]和X线照射组[(35.84±0.52)%](P<0.05),干扰组和X线照射组明显低于空白对照组和阴性对照组(P<0.05)。结论靶向survivin的siRNA能特异性沉默survivin基因表达,诱导细胞凋亡,抑制细胞增殖,进而增强人食管癌Eca-109细胞的放射敏感性。
Objective To investigate the effect of RNAi silencing survivin gene on the radiosensitivity of esophageal cancer Eca-109 cells. Methods Eukaryotic expression plasmid pRNAT-siRNA-survivin was transfected into esophageal cancer Eca-109 cells to construct a negative control plasmid pRNAT-siRNA-control Eca-109 cells were negative control group, untransfected Eca-109 cells were blank control group, and Western blot was used to detect the expression of survivin protein in the three groups. The Eca-109 / si-survivin cells in the logarithmic growth phase interference group and the Eca-109 cells in the blank control group were given 6 Gy of X-ray irradiation under the linear accelerator respectively, and were respectively interference + X-ray irradiation group and simple X-ray irradiation group (X-ray irradiation group). The apoptosis index of each group was detected by flow cytometry. Cell viability was analyzed by MTT assay. Results The expression of survivin protein in the interference group was significantly lower than that in the negative control group (44.17 ± 3.15) and the blank control group (46.20 ± 2.62) (P <0.05). The apoptosis index [ (29.56 ± 0.74)%] was significantly higher than that of the blank control group [(4.35 ± 0.15)%], the negative control group (4.32 ± 0.32)%, the interference group (9.24 ± 0.35)% and the X-irradiation group (22.17 ± 0.75)%], the differences were statistically significant (P <0.05), the interference group and X-ray irradiation group was significantly higher than the blank control group and the negative control group (P <0.05) (19.54 ± 1.12)%] was significantly lower than that of the control group [(95.35 ± 1.40)%], the negative control group (93.45 ± 1.38%), the interference group (70.96 ± 0.85)% and the X-ray (35.84 ± 0.52)%] (P <0.05), and the interference group and the X-ray irradiation group were significantly lower than the blank control group and the negative control group (P <0.05). Conclusion siRNA targeting survivin can silence the expression of survivin gene specifically, induce cell apoptosis, inhibit cell proliferation and further enhance the radiosensitivity of human esophageal cancer Eca-109 cells.