| ZHAO Huan-yu,ZHANG Wei-ming,CHEN Jin-fei.Effects of X-ray irradiation combined with RNAi against STAT3 on radiosensitivity of human esophageal carcinoma cells[J].Chinese Journal of Radiological Medicine and Protection,2011,31(2):180-184 |
| Effects of X-ray irradiation combined with RNAi against STAT3 on radiosensitivity of human esophageal carcinoma cells |
| Received:January 26, 2010 |
| DOI：10.3760/cma.j.issn.0254-5098.2011.02.016 |
| KeyWords:Radiation RNA interference STAT3 gene Esophageal carcinoma Radiosensitivity |
| FundProject:南京医科大学科技发展基金项目(09NJMUZ33) |
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| Abstract:: |
| 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 Eca-109 cells. |
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