Dan Chao, Zhu Heng-cheng, Liu Xiu-heng, Yao Qi-sheng
Department of Urology, Taihe Hospital, Hubei Medical University, Shiyan 442000, China.
Department of Urology, Renmin Hospital, Wuhan University, Wuhan, Hubei 430060, China. Email:
Zhonghua Yi Xue Za Zhi. 2013 Nov 12;93(42):3355-9.
To explore the transfecting effects of RelB small interfering RNA (RelB-siRNA) on murine prostate cancer cell line RM-1 sensitivity of radiotherapy.
The RM-1 cells were divided into RelB-siRNA vector, empty vector, non-transfection and normal groups. After transfection, the first three groups were irradiated by X ray. Clone formation array method was used to measure the surviving fraction and the relevant parameter values after 0, 2, 4, 6, 8 Gy irradiation. Apoptotic rate was measured via Annexin V/PI flow cytometry after 6 Gy irradiation. The level of RelBmRNA was estimated by real-time polymerase chain reaction (PCR) after 6 Gy irradiation. And the RelB protein of cytoplasm and nucleus was detected by Western blot after 6 Gy irradiation.
Clone formation array showed that RelB group at each dose point corresponding to the survival fraction were lower that the empty vector nd non-transfection groups. The D0, Dq and SF2 values of RelB group were 1.68, 0.60, 0.43, lower than those of empty vector group 1.92, 3.08, 0.89 and non-transfection group 1.93, 2.76, 0.84. Annexin V/PI flow cytometry demonstrated that the apoptotic rate of RelB group was 15.27% ± 1.62% and it was significantly higher than the non-transfection group 7.90% ± 1.50% and the empty vector group 8.40% ± 0.69% respectively (P < 0.01) .Real-time PCR indicated that RelB-mRNA amplification of RelB group was 1.18 ± 0.03 and it was significantly lower than the non-transfection group 2.10 ± 0.61 and the empty vector group 1.97 ± 0.66 respectively (P < 0.01) .Western blot suggested that cytoplasmic gray-scale ratio of RelB group was 0.50 ± 0.08 and it was significantly lower than the non-transfection 1.77 ± 0.19 and the empty group 1.52 ± 0.12 respectively (P < 0.01) . And the nuclear gray-scale radio of the RelB group was 0.18 ± 0.03 and it was significantly lower than the non-transfection group 0.61 ± 0.12 and the empty group 0.54 ± 0.13 respectively (P < 0.01) . The RelB protein inhibition rates of cytoplasmic and nuclear RelB-siRNA were 71.8% and 70.4% respectively.
RelB-siRNA can effectively inhibit the RelB expression of murine prostate cancer cell line RM-1 and significantly increase its sensitivity to radiotherapy.
探讨RelB小干扰RNA（RelB-siRNA）对小鼠前列腺癌细胞系RM-1放疗敏感性的转染效果。
将RM-1细胞分为RelB-siRNA载体组、空载体组、未转染组和正常组。转染后，前三组进行X线照射。采用克隆形成实验法测量0、2、4、6、8 Gy照射后细胞存活分数及相关参数值。6 Gy照射后通过Annexin V/PI流式细胞术检测凋亡率。6 Gy照射后通过实时聚合酶链反应（PCR）评估RelBmRNA水平。6 Gy照射后通过蛋白质免疫印迹法检测细胞质和细胞核中的RelB蛋白。
克隆形成实验显示，RelB组各剂量点对应的存活分数均低于空载体组和未转染组。RelB组的D0、Dq和SF2值分别为1.68、0.60、0.43，低于空载体组的1.92、3.08、0.89和未转染组的1.93、2.76、0.84。Annexin V/PI流式细胞术表明，RelB组的凋亡率为15.27%±1.62%，分别显著高于未转染组的7.90%±1.50%和空载体组的8.40%±0.69%（P<0.01）。实时PCR表明，RelB组的RelB-mRNA扩增倍数为1.18±0.03，分别显著低于未转染组的2.10±0.61和空载体组的1.97±0.66（P<0.01）。蛋白质免疫印迹法提示，RelB组细胞质灰度比值为0.50±0.08，分别显著低于未转染组的1.77±0.19和空载体组的1.52±0.12（P<0.01）。RelB组细胞核灰度比值为0.18±0.03，分别显著低于未转染组的0.61±0.12和空载体组的0.54±0.13（P<0.01）。细胞质和细胞核中RelB-siRNA对RelB蛋白的抑制率分别为71.8%和70.4%。
RelB-siRNA可有效抑制小鼠前列腺癌细胞系RM-1中RelB的表达，并显著提高其放疗敏感性。
