Zhang Song-An, Niyazi Hu-Er-Xi-Dan, Hong Wen, Tuluwengjiang Gu-Li-Xian, Zhang Lei, Zhang Yang, Su Wei-Peng, Bao Yong-Xing
1 Cancer Centre, The First Affiliated Hospital of Xinjiang Medical University, Urumqi, P.R. China.
2 Anus-Intestines Department, The First Affiliated Hospital of Xinjiang Medical University, Urumqi, P.R. China.
Tumour Biol. 2017 Mar;39(3):1010428317692237. doi: 10.1177/1010428317692237.
This study aimed to investigate the effect of EBI3 on radiation-induced immunosuppression of cervical cancer HeLa cells by regulating Treg cells through PD-1/PD-L1 signaling pathway. A total of 43 adult female Wistar rats were selected and injected with HeLa cells in the caudal vein to construct a rat model of cervical cancer. All model rats were randomly divided into the radiotherapy group ( n = 31) and the control group ( n = 12). The immunophenotype of Treg cells was detected by the flow cytometry. The protein expressions of EBI3, PD-1, and PD-L1 in cervical cancer tissues were tested by the streptavidin-peroxidase method. HeLa cells in the logarithmic growth phase were divided into four groups: the blank, the negative control group, the EBI3 mimics group, and the EBI3 inhibitors group. Western blotting was used to detect PD-1 and PD-L1 protein expressions. MTT assay was performed to measure the proliferation of Treg cells. Flow cytometry was used to detect cell cycle and apoptosis, and CD4/CD8 T cell ratio in each group. Compared with before and 1 week after radiotherapy, the percentages of CD4T cells and CD8T cells were significantly decreased in the radiotherapy group at 1 month after radiotherapy. Furthermore, down-regulation of EBI3 and up-regulation of PD-1 and PD-L1 were observed in cervical cancer tissues at 1 month after radiotherapy. In comparison to the blank and negative control groups, increased expression of EBI3 and decreased expressions of PD-1 and PD-L1 were found in the EBI3 mimics group. However, the EBI3 inhibitors group had a lower expression of EBI3 and higher expressions of PD-1 and PD-L1 than those in the blank and negative control groups. The EBI3 mimics group showed an increase in the optical density value (0.43 ± 0.05), while a decrease in the optical density value (0.31 ± 0.02) was found in the EBI3 inhibitors group. Moreover, compared with the blank and negative control groups, the apoptosis rates of Treg/CD4T/CD8T cells were decreased in the EBI3 mimics group, but the EBI3 inhibitors group exhibited an increase in apoptosis rate. In conclusion, over-expression of EBI3 could reduce the apoptosis of Treg/CD4T/CD8T cells and prevent radiation-induced immunosuppression of cervical cancer HeLa cells by inhibiting the activation of PD-1/PD-L1 signaling pathway.
本研究旨在通过PD-1/PD-L1信号通路调节调节性T细胞（Treg细胞），探讨EBI3对宫颈癌HeLa细胞辐射诱导免疫抑制的影响。选取43只成年雌性Wistar大鼠，尾静脉注射HeLa细胞构建宫颈癌大鼠模型。将所有模型大鼠随机分为放疗组（n = 31）和对照组（n = 12）。采用流式细胞术检测Treg细胞的免疫表型。采用链霉亲和素-过氧化物酶法检测宫颈癌组织中EBI3、PD-1和PD-L1的蛋白表达。将对数生长期的HeLa细胞分为四组：空白组、阴性对照组、EBI3模拟物组和EBI3抑制剂组。采用蛋白质印迹法检测PD-1和PD-L1蛋白表达。采用MTT法检测Treg细胞的增殖情况。采用流式细胞术检测各组细胞周期、凋亡情况及CD4/CD8 T细胞比值。与放疗前及放疗后1周相比，放疗组放疗后1个月CD4T细胞和CD8T细胞百分比显著降低。此外，放疗后1个月宫颈癌组织中EBI3表达下调，PD-1和PD-L1表达上调。与空白组和阴性对照组相比，EBI3模拟物组EBI3表达增加，PD-1和PD-L1表达降低。然而，EBI3抑制剂组EBI3表达低于空白组和阴性对照组，PD-1和PD-L1表达高于空白组和阴性对照组。EBI3模拟物组光密度值升高（0.43±0.05），而EBI3抑制剂组光密度值降低（0.31±0.02）。此外，与空白组和阴性对照组相比，EBI3模拟物组Treg/CD4T/CD8T细胞凋亡率降低，而EBI3抑制剂组凋亡率升高。综上所述，EBI3过表达可降低Treg/CD4T/CD8T细胞凋亡，通过抑制PD-1/PD-L1信号通路激活预防宫颈癌HeLa细胞辐射诱导的免疫抑制。
