HOXD10在胆管癌细胞放疗增敏中的作用

Role of HOXD10 in radiosensitivity of cholangiocarcinoma cells

目的 :探讨同源异型盒D10(homeobox D10,HOXD10)基因在人胆管癌细胞放疗增敏中的作用及其可能的作用机制。方法:用不同剂量的放射线照射胆管癌RBE和HCCC9810细胞后,应用实时荧光定量PCR法检测细胞中HOXD10 mRNA的表达。应用克隆形成实验、"多靶单击模型"和FCM法检测HOXD10稳定过表达的胆管癌LvHOXD10-RBE和Lv-HOXD10-HCCC9810细胞的放射增敏效应及细胞的凋亡情况,蛋白质印迹法检测放射治疗对HOXD10过表达的胆管癌LvHOXD10-RBE和Lv-HOXD10-HCCC9810细胞中凋亡相关蛋白表达的影响。结果:2、4和8 Gy放射治疗后48和72 h,胆管癌RBE和HCCC9810细胞中HOXD10 mRNA的表达水平均显著高于放疗后24 h组(P值均<0.05)。在2、4和8 Gy放射治疗后,Lv-HOXD10-RBE和LvHOXD10-HCCC9810细胞的克隆形成率均分别显著低于对应的仅表达绿色荧光蛋白的阴性对照Lv-CTRL-RBE和Lv-CTRL-HCCC9810细胞(P值均<0.05);Lv-HOXD10-RBE和Lv-HOXD10-HCCC9810细胞的平均致死剂量(mean lethal dose,D0)、准阈剂量(quasithreshold dose,Dq)和2 Gy单次照射后细胞的存活分数(survival fraction with 2 Gy,SF2)值均分别低于Lv-CTRL-RBE和Lv-CTRL-HCCC9810细胞(P值均<0.05)。4 Gy放射治疗后24 h,Lv-HOXD10-RBE和Lv-HOXD10-HCCC9810细胞凋亡率高于Lv-CTRL-RBE和Lv-CTRL-HCCC9810细胞(P值均<0.05);Lv-HOXD10-RBE和Lv-HOXD10-HCCC9810细胞中p53、p21和Bax蛋白的表达水平均高于Lv-CTRL-RBE和Lv-CTRLHCCC9810细胞,而双微体2(murine double minute 2,Mdm2)蛋白的表达水平均低于Lv-CTRL-RBE和Lv-CTRL-HCCC9810细胞(P值均<0.05)。结论:HOXD10可增加人胆管癌RBE和HCCC9810细胞对放射的敏感性,这一作用可能与凋亡相关蛋白的表达上调有关。

Objective： To investigate the role of homeobox D10 （HOXD10） in radiosensitivity of cholangiocarcinoma cells and its possible mechanism. Methods： The expression of HOXDIO mRNA in cholangiocarcinoma RBE and HCCC9810 cells after X-ray irradiation was detected by real-time fluorescent quantitative PCR. The radiosensitivity and apoptosis of cholangiocarcinoma Lv-HOXD10-RBE and Lv-HOXD10- HCCC9810 cells with stable overexpression of HOXD10 after irradiation were evaluated by colony formation assay, multi-target single-hit model and FCM, respectively. The expression levels of apoptosis-related proteins in cholangiocarcinoma cells with stable overexpression of HOXD10 after irradiation were detected by Western blotting. Results＂ The expressions of HOXD10 mRNA in RBE and HCCC9810 cells after 2-, 4- and 8-Gy X-ray irradiation for 48 and 72 h were significantly higher than those receiving irradiation for 24 h （all P 〈 0.05）. The colony formation rates of Lv-HOXDIO-RBE and Lv-HOXD10- HCCC9810 cells after 2, 4 and 8 Gy X-ray irradiation were significantly lower than those of the Lv-CTRL-RBE and Lv-CTRL-HCCC9810 cells only expressing green fluorescent protein as the negative controls （all P 〈 0.05）. The values of mean lethal dose （D0）, quasithreshold dose （Dq） and survival fraction with 2 Gy （SF2） of Lv-HOXD10-RBE and Lv-HOXD10-HCCC9810 cells were significantly lower than those of Lv-CTRL-RBE and Lv-CTRL-HCCC9810 cells （all P 〈 0.05）. The apoptotic rates of Lv-HOXD10-RBE and Lv-HOXD10-HCCC9810 cells after 8 Gy irradiation for 24 h were higher than those of the Lv-CTRL-RBE and Lv-CTRL-HCCC9810 cells （all P 〈 0.05）. The expression levels of p53, p21 and Bax proteins in Lv-HOXD10-RBE and Lv-HOXD10-HCCC981 0 cells after irradiation were higher than those in the Lv-CTRL-RBE and Lv-CTRL-HCCC9810 cells, and the expression level of murine double minute 2 （Mdm2） was opposite （all P 〈 0.05）. Conclusion： HOXD10 can increase the radiosensitivity of cholangiocarcinoma RBE and HCCC9810 cells. This effect may be related to up-relagulation of apoptosis-related proteins.