X射线照射联合RNA干扰STAT3基因对人食管癌细胞放射敏感性的影响

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

目的探讨X射线照射联合RNA干扰STAT3基因对人食管癌细胞放射敏感性的影响。方法针对STAT3mRNA序列，设计合成3对编码小于扰RNA（siRNA）的DNA模板寡核苷酸链（siRNAl、siRNA2和siRNA3），构建STAT3-siRNA阳性和阴性重组质粒，用以转染Eca-109细胞。Eca-109细胞分为5组：转染试剂对照组、阴性质粒对照组、重组质粒siRNAl实验组、siRNA2实验组和siRNA3实验组。细胞转染后48h，换用Gdl8培养液筛选21d，获得单个阳性克隆并扩增。分别用RT-PCR和Westernblotting检测细胞中的STAT3mRNA和蛋白表达。转染细胞分别接受0、2、4、6和8Gyx射线照射，平板克隆形成实验检测细胞存活分数，流式细胞仪检测经4GyX射线照射的细胞周期和凋亡。结果成功构建了STAT3-siRNA3重组质粒。RT·PCR和Westernblotting显示，siRNA3实验组的STAT3mRNA和蛋白表达显著少于2个对照组，而siRNAl和siRNA2实验组的STAT3mRNA和蛋白表达与对照组比较差异无统计学意义。在2～8Gy剂量点，siRNA3实验组细胞存活分数均显著低于对照组（t=-0．228～-0．051，P〈0.05）。流式细胞仪分析显示，经4Gyx射线照射，siRNA3实验组G0／G，期细胞的百分率和细胞凋亡率均显著高于转染试剂对照组、阴性质粒对照组（t=-13.137～16.350，P〈0.01），4Gyx射线照射可引起细胞周期G0／G1期阻滞，诱导肿瘤细胞凋亡。结论STAT3-siRNA3能有效地抑制肿瘤细胞的STAT3mRNA和蛋白表达。4GyX射线照射联合STAT3-siRNA3可抑制人食管癌细胞增殖，引起细胞周期阻滞和凋亡，提高肿瘤细胞的放射敏感性。

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. I-siRNA3 ） , and a STAT3-siRNA negative recombinant plasmid （ pRNAT-U6. 1-negative） were thus constructed and then transfeeted into the cultured Eca-109 cells, which were divided into transfectiou reagent control group, pRNAT-U6. 1-siRNA1-3 transfection groups, and pRNAT-U6, l-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 ceils 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. I-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 controlgroups. 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/G~ 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.