Department of Urology, First Affiliated Hospital of Soozhow University, Suzhou, Jiangsu, China.
Urology. 2012 Mar;79(3):744.e17-24. doi: 10.1016/j.urology.2011.10.024. Epub 2011 Dec 22.
To evaluate the effect of silencing hypoxia-inducible factor-1α (HIF-1α) expression by small interfering RNA (siRNA) on the radiosensitivity of the PC3 cell line.
The expression of HIF-1α in PC3, a p53-null and androgen-independent prostate cancer cell line, was knocked down by siRNA. Irradiation was performed at 48 hours after transfection. The cells were divided into 3 groups: the PC3 group, control group (transfected with scramble siRNA), and HIF-1α silence group. HIF-1α expression was determined using real-time polymerase chain reaction and Western immunoblotting. A clonogenic assay and the cell counting kit-8 assay were performed to determine the radiosensitivity. Flow cytometry was used to assess apoptosis and cell cycle distribution.
HIF-1α siRNA downregulated HIF-1α expression in PC3 cells on the mRNA level and protein level, and its silencing effect on mRNA level was evident at 24-72 hours. The HIF-1α silence group had a low final slope of exponential part of a radiation survival curve, survival fraction of 2 Gy, quasi-threshold dose, and extrapolation number, and the sensitizing enhancement ratio was 1.24. The cell counting kit-8 assay showed decreased cellular viability (24 hours, F = 139.74, P < .01; 48 hours, F = 495.49, P < .01; 72 hours, F = 426.89, P < .01; 96 hours, F = 471.11, P < .01) in the HIF-1α silence group. Silencing HIF-1α also induced more apoptosis (PC3, 17.9% ± 1.65%; control group, 18.6% ± 1.37%; HIF-1α silence group, 29.1% ± 2.16%; F = 169.9, P < .01) and cell cycle arrest at the S, G(2)/M phase.
The suppression of HIF-1α in PC3 cells sensitizes the PC3 cells to irradiation. We have shown that HIF-1α inhibition attenuates repair of postradiation injury, with an increase in both interphase death and reproductive death after irradiation, apoptotic potential, and cell cycle arrest at the proliferative phase.
通过小干扰 RNA（siRNA）沉默缺氧诱导因子-1α（HIF-1α）的表达，评估其对 PC3 细胞系放射敏感性的影响。
用 siRNA 敲低 p53 缺失和雄激素非依赖性前列腺癌细胞系 PC3 中 HIF-1α 的表达。转染后 48 小时进行照射。将细胞分为 3 组：PC3 组、对照组（转染 scramble siRNA）和 HIF-1α 沉默组。使用实时聚合酶链反应和 Western 免疫印迹法测定 HIF-1α 的表达。通过集落形成实验和细胞计数试剂盒-8 实验测定放射敏感性。流式细胞术评估细胞凋亡和细胞周期分布。
HIF-1α siRNA 在 PC3 细胞中下调 HIF-1α 的 mRNA 和蛋白表达，其在 24-72 小时对 mRNA 水平的抑制作用明显。HIF-1α 沉默组的放射生存曲线指数部分的最终斜率低、2 Gy 时的存活率、准阈值剂量和外推数较低，增敏比为 1.24。细胞计数试剂盒-8 实验显示，HIF-1α 沉默组细胞活力降低（24 小时，F = 139.74，P <.01；48 小时，F = 495.49，P <.01；72 小时，F = 426.89，P <.01；96 小时，F = 471.11，P <.01）。沉默 HIF-1α 还诱导更多的细胞凋亡（PC3 为 17.9%±1.65%；对照组为 18.6%±1.37%；HIF-1α 沉默组为 29.1%±2.16%；F = 169.9，P <.01）和 S、G2/M 期的细胞周期阻滞。
PC3 细胞中 HIF-1α 的抑制可使 PC3 细胞对照射敏感。我们已经表明，HIF-1α 抑制可减弱照射后损伤的修复，增加照射后有丝分裂死亡和生殖死亡、凋亡潜能以及增殖期的细胞周期阻滞。
