Zhang Heping, Li Jiancheng, Cheng Wenfang, Liu D I, Chen Cheng, Wang Xiaoying, Lu Xujing, Zhou Xifa
Department of Radiation Oncology, Changzhou Tumor Hospital of Soochow University, Changzhou, Jiangsu 213002, P.R. China.
Department of Radiation Oncology, Fujian Provincial Tumor Hospital, Provincial Clinical College of Fujian Medical University, Fuzhou, Fujian 350014, P.R. China.
Oncol Lett. 2015 Sep;10(3):1495-1500. doi: 10.3892/ol.2015.3456. Epub 2015 Jul 3.
The present study investigated the effects of small interfering RNAs (siRNAs) specific to the epidermal growth factor receptor (EGFR) gene, on the radiosensitivity of esophageal squamous cell carcinoma cells. EGFR gene siRNAs (EGFR-siRNA) were introduced into esophageal cancer Eca109 cells using Lipofectamine® 2000. The EGFR messenger (m)RNA expression levels, EGFR protein expression and cell growth were assessed using reverse transcription-polymerase chain reaction analysis, western blot analysis and a Cell Counting Kit-8 (CCK-8), respectively. In addition, colony assays were used to determine the inhibitory effects of X-ray radiation on EGFR-silenced cells. EGFR mRNA and protein levels were reduced in the Eca109 cells transfected with EGFR-siRNA. The relative EGFR mRNA expression levels were reduced to 26.74, 9.52 and 4.61% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These mRNA levels were significantly reduced compared with the those of the control group (42.44%; P<0.0001). Transfection with siRNA3 resulted in the greatest reduction in EGFR mRNA expression, with an inhibition rate of 85%. The relative EGFR protein expression levels were reduced to 24.05, 34.91 and 34.14% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These protein levels were significantly reduced compared with those of the control group (78.57%; P<0.0001). Transfection with siRNA1 resulted in the greatest reduction in EGFR protein expression, with an inhibition rate of 72.84%. This reduction in EGFR expression inhibited the proliferation of Eca109 cells, which was identified using the CCK-8 assay. The proliferation inhibition ratio was 28.2%. The cells treated with irradiation in addition to EGFR-siRNA, demonstrated reduced radiobiological parameters (D0, Dq and SF2) compared with those of cells treated with irradiation only, with a sensitization enhancing ratio of 1.5. In conclusion, suppression of EGFR expression may enhance the radiosensitivity of esophageal cancer Eca109 cells and therefore may represent a promising approach for future clinical practice.
本研究调查了表皮生长因子受体（EGFR）基因特异性小干扰RNA（siRNA）对食管鳞状细胞癌细胞放射敏感性的影响。使用Lipofectamine® 2000将EGFR基因siRNA（EGFR-siRNA）导入食管癌Eca109细胞。分别采用逆转录-聚合酶链反应分析、蛋白质印迹分析和细胞计数试剂盒-8（CCK-8）评估EGFR信使（m）RNA表达水平、EGFR蛋白表达和细胞生长情况。此外，采用集落形成试验确定X射线辐射对EGFR沉默细胞的抑制作用。用EGFR-siRNA转染的Eca109细胞中EGFR mRNA和蛋白水平降低。用EGFR-siRNA1、2和3转染的Eca109细胞中，相对EGFR mRNA表达水平分别降至26.74%、9.52%和4.61%。与对照组（42.44%）相比，这些mRNA水平显著降低（P<0.0001）。用siRNA3转染导致EGFR mRNA表达降低最多，抑制率为85%。用EGFR-siRNA1、2和3转染的Eca109细胞中，相对EGFR蛋白表达水平分别降至24.05%、34.91%和34.14%。与对照组（78.57%）相比，这些蛋白水平显著降低（P<0.0001）。用siRNA1转染导致EGFR蛋白表达降低最多，抑制率为72.84%。这种EGFR表达的降低抑制了Eca109细胞的增殖，这是通过CCK-8试验确定的。增殖抑制率为28.2%。与仅接受辐射处理的细胞相比，除EGFR-siRNA外还接受辐射处理的细胞显示放射生物学参数（D0、Dq和SF2）降低，增敏增强比为1.5。总之，抑制EGFR表达可能增强食管癌Eca109细胞的放射敏感性，因此可能是未来临床实践中一种有前景的方法。
