Objective: To explore the specific role and molecular mechanism of octamer-binding transcription factor 4 (Oct4) in promoting the progression of esophageal squamous cell carcinoma and radioresistance. Methods: The Gene Expression Profile Data Dynamic Analysis (GEPIA) database was used to analyze the expression differences of the Oct4 gene in different types of tumor tissues and their corresponding adjacent normal tissues. The clinical data and surgical resection tissue specimens of 196 patients with esophageal squamous cell carcinoma who received surgery combined with radiotherapy at Henan Provincial Chest Hospital from January 2013 to May 2022 were collected. Immunohistochemistry was used to detect the expression of Oct4 protein in the tumor and adjacent tissues. The lentiviral packaging system was used to construct esophageal squamous cell carcinoma cell lines that up-regulated or down-regulated Oct4. The cell counting kit 8 (CCK-8) was used to detect the cell proliferation ability, the scratch test was used to detect the cell migration ability, and the clone formation test was used to detect the cell radiosensitivity. Immunofluorescence experiment was used to detect DNA damage level, and Western blot was used to detect the expressions of Oct4, human phosphorylated histone (γ-H2AX), E-cadherin, N-cadherin, vimentin, and zinc finger E box binding homology box 1 (ZEB1). Results: The analysis of GEPIA database showed that the expression level of Oct4 mRNA in esophageal carcinoma was higher than that in paracancerous tissues. The expression level of Oct4 protein in tumor tissues was 78.35±1.42, which was higher than that in adjacent tissues (16.27±0.49). The survival time of patients with a high expression of Oct4 was significantly shorter than that of patients with a low expression of Oct4 (25.40 and 47.00 months). Compared with the control group, the proliferation ability of KYSE510 cells in the Oct4 up-regulated group was enhanced after 72-h culture, and the cell migration ability of these cells was also enhanced, with the migration rate being (41.67±1.20)% vs (23.67±1.86)% after 24-h culture. The radiosensitivity of cells in this group decreased, with the radiosensitivity enhancement ratio being 0.69±0.06 vs 1.00±0.02. After radiotherapy, the expressions of γ-H2AX and E-cadherin decreased, while the expressions of ZEB1, vimentin and N-cadherin increased. Compared with the control group, the proliferation ability of KYSE150 cells in the Oct4 down-regulated groups 1 and 2 decreased (absorbance being 2.51±0.17, 2.38±0.16, and 3.33±0.07, respectively, P＜0.01) after 72-h culture, and the migration ability also decreased, with the migration rate being (13.33±0.88)%, (13.00±1.00)%, and (40.33±2.03)%, respectively (all P＜0.001), after 24-h culture. The radiosensitivity was enhanced, with the radiosensitivity enhancement ratio being 1.34±0.11,1.24±0.07, and 1.00±0.02, respectively (all P＜0.05). After radiotherapy, the expressions of γ-H2AX and E-cadherin increased, while the expressions of ZEB1, vimentin and N-cadherin decreased. Compared with the control group, the proliferation ability of KYSE510 cells in the ZEB1 down-regulated group decreased [absorbance being 1.33±0.15 vs 1.81±0.16 (P=0.002)] after 72-h culture. The radiosensitivity was enhanced, with the radiosensitivity enhancement ratio being 1.37±0.11 vs 1.00±0.01 (P=0.037), and after radiotherapy the expression of γ-H2AX increased. Conclusion: Oct4 is involved in the regulation of epithelial-mesenchymal transformation of esophageal squamous cell carcinoma, which promotes the proliferation, migration, and radioresistance of esophageal squamous cell carcinoma.
目的： 探讨八聚体结合转录因子4（Oct4）与食管鳞状细胞癌进展和放疗抵抗的具体作用和分子机制。 方法： 利用基因表达谱数据动态分析（GEPIA）数据库，分析Oct4基因在不同类型肿瘤组织和对应癌旁正常组织中的表达差异。2013年1月至2022年5月在河南省胸科医院接受手术联合放疗治疗的196例食管鳞状细胞癌患者，收集其临床资料及手术切除组织标本，采用免疫组化法检测癌及癌旁组织中Oct4蛋白的表达。通过慢病毒包装系统构建上调和下调Oct4的食管鳞状细胞癌细胞系，采用细胞计数试剂盒8法检测细胞增殖能力，划痕实验检测细胞迁移能力，克隆形成实验检测细胞的放疗敏感性，免疫荧光实验检测DNA损伤水平，Western blot检测Oct4、人磷酸化组蛋白（γ-H2AX）、E-cadherin、N-cadherin、vimentin、锌指E盒结合同源盒1（ZEB1）的表达。 结果： GEPIA数据库资料分析显示，食管癌组织中Oct4 mRNA的表达水平高于其癌旁组织（P＜0.05）。196例食管鳞状细胞癌患者的肿瘤组织中Oct4蛋白表达水平为78.35±1.42，高于癌旁组织（16.27±0.49，P＜0.001）。Oct4高表达组患者的生存时间明显短于Oct4低表达组（中位生存时间分别为25.40和47.00个月；HR=1.68，P=0.022）。与对照组比较，Oct4上调组KYSE510细胞增殖能力增强（培养72 h，吸光度分别为1.73±0.19和1.35±0.10，P=0.007），迁移能力增强［培养24 h，细胞迁移率分别为（41.67±1.20）%和（23.67±1.86）%，P=0.001］，放疗敏感性降低（辐射增敏比分别为0.69±0.06和1.00±0.02，P=0.010），放疗后γ-H2AX表达水平降低，ZEB1、vimentin和N-cadherin表达升高，E-cadherin表达下降；Oct4下调1组和Oct4下调2组KYSE150细胞增殖能力减弱（培养72 h，吸光度分别为2.51±0.17、2.38±0.16和3.33±0.07，均P＜0.01），迁移能力下降［培养24 h，细胞迁移率分别为（13.33±0.88）%、（13.00±1.00）%和（40.33±2.03）%，均P＜0.001］，放疗敏感性增强（辐射增敏比分别为1.34±0.11、1.24±0.07和1.00±0.02，均P＜0.05），放疗后γ-H2AX表达水平升高，ZEB1、vimentin和N-cadherin表达下降，E-cadherin表达升高。与对照组比较，ZEB1下调组KYSE510细胞增殖能力减弱（培养72 h，吸光度分别为1.33±0.15和1.81±0.16，P=0.002），放疗敏感性增强（辐射增敏比分别为1.37±0.11和1.00±0.01，P=0.037），放疗后γ-H2AX表达水平升高。 结论： Oct4参与调节食管鳞状细胞癌细胞上皮-间质转化，进而促进食管鳞状细胞癌细胞增殖、迁移和放疗抵抗。.