RNA interference for epidermal growth factor receptor enhances the radiosensitivity of esophageal squamous cell carcinoma cell line Eca109

Abstract

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.

Keywords: cell lines; epidermal growth factor receptor; esophageal neoplasm; radiosensitivity; small interfering RNA.

Figure 1.

Eca109 cells successfully transfected with small interfering RNA 3. (A) Fluorescent imaging of Cy3 reporter (red) post-transfection, 200 bright fields. (B) Fluorescent microscope image post-transfection, 200 dark fields.

Figure 2.

EGFR-siRNA transfection reduces EGFR mRNA expression levels. (A) Reverse transcription-PCR was used to detect the EGFR and β-actin gene expression in the various groups 48 h following transfection. (B) PCR products were semi-quantified by assessing the relative intensity of messenger RNA expression using image analysis by comparing EGFR with β-actin. Data are presented as the mean ± standard deviation of three independent experiments (*P<0.0001 vs. Control). EGFR, epidermal growth factor receptor; siRNA, small interfering RNA; PCR, polymerase chain reaction; NC, non-targeting siRNA (negative control); PC, postitive interference siRNA (positive control); Control, blank control group (untransfected).

Figure 3.

EGFR-siRNA treatment reduces EGFR protein expression. (A) EGFR and β-tubulin protein expression in the various groups was detected by western blotting. (B) Semi-quantitative analysis of the EGFR protein expression comparing EGFR to β-tubulin. Data are presented as the mean ± standard deviation of three experiments; *P<0.0001 vs. Control). NC, negative interference siRNA; PC, positive interference siRNA; Control, blank control group (untransfected); EGFR, epidermal growth factor receptor; siRNA, small interfering RNA.

Figure 4.

EGFR-siRNA treatment inhibits Eca109 cell proliferation asdetermined by CCK-8 assay. OD values of Eca109 cell proliferation following transfection with EGFR-siRNA. Results are presented as mean ± standard deviation of four separate experiments (*P<0.05 vs. Control). NC, negative interference siRNA; PC, positive interference siRNA; Control, blank control group (untransfected); EGFR, epidermal growth factor receptor; siRNA, small interfering RNA; OD, optical density.

Figure 5.

EGFR-siRNA increases the radiosensitivity of Eca109 cells. Survival curve of EGFR-siRNA and control Eca109 cells treated with various doses of X-ray irradiation. EGFR, epidermal growth factor receptor; siRNA, small interfering RNA.