Zhang Rui, Han Dan, Li Lu, Luo Wenguang, Liu Jing, Qian Liting
Department of Oncology, QingPu Branch of Zhongshan Hospital Affiliated to Fudan University, Shanghai 201799, People's Republic of China.
Division of Life Science and Medicine, University of Science and Technology of China, Hefei, Anhui 230001, People's Republic of China.
Cancer Manag Res. 2020 Oct 2;12:9539-9549. doi: 10.2147/CMAR.S261182. eCollection 2020.
Radiotherapy is one of the most important treatments for esophageal squamous cell carcinoma (ESCC). Previously, we found that EphA5 expression was increased in ESCC cells and tumor tissues. Studies from other groups reported that EphA5 is abnormally expressed in numerous malignant tumors and may be involved in the radiosensitivity of lung cancer. However, the role of EphA5 in radiotherapy for ESCC remains unclear.
The siRNA sequences against human were transfected to the ESCC cells (KYSE150 and KYSE450). After ionizing radiation (IR), cell viability and colony formation assays were used to test the changes of cell proliferation in EphA5-silenced cells. Flow cytometry analysis was performed to investigate the cell apoptosis and cycle in the irradiated cells interfered by siRNA. The key molecules involved in cell cycle checkpoints and DNA damage repair were evaluated by Western blot and immunofluorescence.
CCK8 assay and clonogenic assay showed that the proliferation of EphA5-silenced ESCC cells was inhibited after IR. At 24 h post-IR treatment, we found that the G1/S checkpoint triggered by DNA damage in EphA5-silenced cells was defective. γ-H2AX foci in the irradiated EphA5-silenced cells were impaired at 0.5 h post-IR treatment as well as ATM activation. The defective activation of ATM resulted in a decrease of p-Chk2, p-p53 and p21 expression.
In conclusion, these results indicate that EphA5 silencing increases radiosensitivity in ESCC cells through ATM-dependent pathway, which provides a potential target for the radiotherapy in ESCC.
放射治疗是食管鳞状细胞癌（ESCC）最重要的治疗方法之一。此前，我们发现EphA5在ESCC细胞和肿瘤组织中的表达增加。其他研究小组报道，EphA5在多种恶性肿瘤中异常表达，可能与肺癌的放射敏感性有关。然而，EphA5在ESCC放射治疗中的作用仍不清楚。
将针对人类EphA5的小干扰RNA（siRNA）序列转染至ESCC细胞（KYSE150和KYSE450）。在进行电离辐射（IR）后，使用细胞活力和集落形成试验检测EphA5沉默细胞中细胞增殖的变化。通过流式细胞术分析研究siRNA干扰后受照射细胞的凋亡和细胞周期。通过蛋白质免疫印迹法和免疫荧光法评估参与细胞周期检查点和DNA损伤修复的关键分子。
CCK8试验和克隆形成试验表明，IR后EphA5沉默的ESCC细胞增殖受到抑制。在IR处理后24小时，我们发现EphA5沉默细胞中由DNA损伤触发的G1/S检查点存在缺陷。在IR处理后0.5小时，受照射的EphA5沉默细胞中的γ-H2AX焦点以及ATM激活受损。ATM的缺陷激活导致p-Chk2、p-p53和p21表达降低。
总之，这些结果表明，EphA5沉默通过ATM依赖途径增加ESCC细胞的放射敏感性，这为ESCC放射治疗提供了一个潜在靶点。
