Background: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. Methods:The siRNA sequences against human EPHA5 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. Results: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. Conclusion: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.

译文

背景:放射治疗是食管鳞状细胞癌(ESCC)的最重要治疗方法之一。以前,我们发现EphA5表达在ESCC细胞和肿瘤组织中增加。其他研究表明,EphA5在许多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放射治疗中的作用尚不清楚。
方法:将针对人类EPHA5的siRNA序列转染至ESCC细胞(KYSE150和KYSE450)。电离辐射(IR)后,细胞活力和集落形成测定法用于测试EphA5沉默的细胞中细胞增殖的变化。进行流式细胞术分析以研究被siRNA干扰的被照射细胞的细胞凋亡和周期。通过蛋白质印迹和免疫荧光评估了参与细胞周期检查点和DNA损伤修复的关键分子。
结果:CCK8法和克隆形成法显示,IR后,EphA5沉默的ESCC细胞的增殖受到抑制。红外线治疗后24小时,我们发现由EphA5沉默的细胞中的DNA损伤触发的G1 / S检查点存在缺陷。照射后的EphA5沉默的细胞中的γ-H2AX焦点在IR处理以及ATM激活后0.5小时受到损害。 ATM激活缺陷导致p-Chk2,p-p53和p21表达降低。
结论:总之,这些结果表明,EphA5沉默可通过ATM依赖性途径提高ESCC细胞的放射敏感性,这为ESCC放射治疗提供了潜在的靶标。

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