Effects of X-ray irradiation combined with RNAi against STAT3 on radiosensitivity of human esophageal carcinoma cells / 中华放射医学与防护杂志

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 euhured.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(siRNAI,siRNA2,and siRNA3),and a negative sequence was synthesized to be used as control.STAT3-siRNA positive recombinant plasmids(pRNAT-U6.1-siRNAI,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-siRNAl-3 transfection groups,and pRNAT-U6.1-negative centrel group.The positive eell clones were screened.RT-PCR and Westem 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 significanfly lower than those of the control groups.At 2-8 Gy, the survival fractions of the siRNA3 group were aU 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.