Effects of X-ray irradiation combined with RNAi against STAT3 on radiosensitivity of human esophageal carcinoma cells

Objective: To explore the effects of X-ray irradiation combined with RNAi against signal transducer and activator of transcription 3 (STAT3) on the radiosensitivity of human esophageal carcinoma cells. Methods: Human esophageal carcinoma cells of the line Eca-109 were cultured. Three pairs of DNA template aiming at the base sequences of the coding regions 2037-2055, 1243-1261, and 455-473 of the STAT3 mRNA were synthesized(siRNA1, siRNA2, and siRNA3), and a negative sequence was synthesized to be used as control. STAT3-siRNA positive recombinant plasmids(pRNAT-U6.1-siRNA1, pRNAT-U6.1-siRNA2, and pRNAT-U6.1-siRNA3), and a STAT3-siRNA negative recombinant plasmid (pRNAT-U6.1-negative) were thus constructed and then transfected into the cultured Eca-109 cells, which were divided into transfection reagent control group, pRNAT-U6.1-siRNA1-3 transfection groups,and pRNAT-U6.1-negative control group. The positive cell clones were screened. RT-PCR and Western blotting were used to detect the STAT3 mRNA and protein expression. The transfected Eca-109 cells were exposed to 0, 2, 4, 6, and 8 Gy of X-rays,respectively, and the survival fraction of the cells was analyzed by clone formation assay. Flow cytometry was applied to analyze the cycle arrest and cell apoptosis 4 Gy post-irradiation. Results: Agarose gel electrophoresis confirmed the successful construction of the plasmid pRNAT-U6.1-siRNA. RT-PCR and Western blotting demonstrated that the mRNA and protein expression levels of STAT3 transfected with STAT3-siRNA3 were both significantly lower than those of the control groups. At 2-8 Gy, the survival fractions of the siRNA3 group were all significantly lowered than those of the control group (t=-0.228 - -0.051, P<0.05). Flow cytometry showed that the percentage of the cell cycle G₀/G₁ phase and the apoptosis rate of the siRNA3 group were both significantly higher than those of the control groups at 4 Gy post-irradiation (t=-13.137-16.350, P<0.01). Conclusions: X-ray irradiation combined with RNAi against STAT3 could inhibit the proliferation of the human esophageal carcinoma cells, induce cell cycle arrest and apoptosis, improve the radiosensitivity in Eta-109 cells. (authors)