PinX1对食管癌细胞放射敏感性及活性氧水平的影响研究

Effect of PinX1 on radiosensitivity and ROS level of esophageal cancer cells

目的研究端粒酶抑制因子X1(PinX1)对食管癌细胞放射敏感性及活性氧(ROS)水平的影响。方法食管癌细胞分为对照组(未做细胞转染)、照射组(8GyX射线)、PinX1组(转染pDsRed1-PinX1至过表达)、照射+PinX1(转染pDsRed1-PinX1至过表达后8GyX射线照射)。MTT测定细胞增殖,流式细胞术测定细胞凋亡,Westernblot测定细胞中含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)和含半胱氨酸的天冬氨酸蛋白水解酶9(Caspase-9)的活化水平,二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)法测定细胞中ROS水平,集落形成实验测定放射敏感性。构建EC9706细胞致裸鼠皮下移植瘤模型,每组20只,小鼠每4天接受5次8Gy剂量的γ射线局部照射,分析异种移植小鼠模型中PinX1过表达与放射敏感性的关系。结果与对照组相比,照射组和PinX1组细胞的存活率从100%降低至(67.92±4.71)%和(83.14±4.01)%,差异有统计学意义(t=9.433、4.957,P<0.05),细胞凋亡率从(6.47±1.46)%升高到(44.38±4.16)%和(25.42±2.78)%,差异有统计学意义(t=12.882、6.439,P<0.05),细胞中Caspase-3活化水平升高(t=6.655、2.531,P<0.05),Caspase-9的活化水平升高(t=3.775、3.088,P<0.05),细胞内ROS水平升高(t=12.110、7.339,P<0.05)。与照射组相比,照射+PinX1组细胞存活率从(67.92±4.71)%下降至(52.73±5.58)%,差异有统计学意义(t=8.942,P<0.05),细胞凋亡率从(44.38±4.16)%升高至(55.29±4.98)%,差异有统计学意义(t=3.707,P<0.05),细胞中Caspase-3活化水平升高(t=15.435,P<0.05),Caspase-9的活化水平也升高,(t=17.990,P<0.05),ROS水平升高(t=4.526,P<0.05)。过表达PinX1的EC9706细胞放射敏感性增加,增敏比为1.408。过表达PinX1的细胞裸鼠移植瘤体积明显减小。结论PinX1抑制食管癌细胞增殖,诱导食管癌细胞凋亡,增加食管癌细胞放射敏感性。

Objective To study the effect of PinX1 on radiosensitivity and ROS level of esophageal cancer cells. Methods Esophageal cancer cells were divided into control group(no cell transfection), radiation group (8 Gy of X-rays), PinX1 group (pDsRed1-PinX1 transfect to overexpression, radiation+ PinX1 group (8 Gy X-rays after pDsRed1-PinX1 transfection). Cell proliferation was measured by MTT assay, cell apoptosis was measured by flow cytometry, the activation levels of Caspase-3 and Caspase-9 in cells were measured by Western blot, the cellular ROS level was determined using DCFH-DA probe, and the cell radiosensitivity was evaluated by the colony formation assay. The subcutaneous xenograft tumor model of EC9706 cells was established with nude mice, and each group received local irradiation every 4 days 8 Gy five fractions γ-rays. The nude mice bearing PinX1-transfected cells were implanted with PinX1 plasmid into tumor before radiotherapy to investigate the relationship between PinX1 overexpression and radiosensitivity in xenotransplantation mice models. Results Compared with control group cells, the survival rate of EC9706 cells of radiation group or PinX1 group decreased significantly from 100% to (67.92±4.71)% and (83.14 ±4.01)% (t=9.433, 4.957, P<0.05), the apoptosis rate increased significantly from(6.47±1.46)% to (44.38±4.16)% and (25.42±2.78)%(t=12.882, 6.439, P<0.05), Caspase-3 activation level increased (t=6.655, 2.531, P<0.05), and the activation level of Caspase-9 and cellular ROS was also significantly increased. Compared with radiation group cells, the cell viability of radiation+ PinX1 group decreased from (67.92 ±4.71)% to (52.73 ±5.58)% (t=8.942, P<0.05), the apoptosis rate increased from (44.38±4.16)% to (55.29±4.98)% (t=3.707, P<0.05), the activation levels of Caspase-3, -9, and the level of ROS in cells was increased (t=15.435, 17.990, 4.526, P<0.05). The radiosensitivity of EC9706 cells was increased after PinX1 transfection with a sensitization ratio of 1.408. The volume of xenograft tumor with PinX1 overexpression cells in nude mice was significantly smaller than control. Conclusions Overexpression of PinX1 could inhibit the proliferation, induce apoptosis and increase the radiosensitivity of esophageal cancer cells.