RNA干扰抑制STAT-1基因表达对食管癌细胞放射生物效应影响

Effect of RNA interference of STAT 1 expression on radiosensitivity and cell cycle of esophageal carcinoma cell line Eca109

目的 利用RNA干扰技术抑制人食管癌细胞（ECA109）信号转导与转录活化因子1（STAT 1）基因表达,观察其对放射敏感性和细胞周期影响。 方法 针对基因STAT 1 设计构建干扰质粒pSTAT 1-shRNA,与慢病毒包装质粒混合后共同转染293T细胞,收集病毒液感染ECA109细胞。采用RT-PCR和蛋白印迹法测定STAT 1在mRNA水平和蛋白水平的表达,采用克隆形成实验和流式细胞术检测ECA109细胞放射敏感性和周期分布。 结果 空白对照、转染阴性、转染阳性细胞的D 0-值分别为2.98、3.02、2.03,SF2分别为0.88、0.88、0.83,Dq值分别为1.39、1.57、1.20。4 Gy照射后12、24、48 h 转染阳性细胞比空白对照和转染阴性细胞的G1G0期比例增高（34.13%∶22.03%∶22.27%、43.80%∶28.40%∶28.63%、53.20%∶42.2%∶41.83%,F=7.56、10.01、10.73,P=0.023、0.012、0.010）和G2＋M期比例降低（14.33%∶32.23%∶32.23%、27.73%∶43.53%∶44.00%、14.23%∶27.97%∶27.93%,F=16.86、26.62、40.34,P=0.003、0.001、0.000）。结论 RNA干扰不影响ECA109细胞的增殖活性,可能是通过调节照射后细胞周期分布来增加放射敏感性。

Objective To inhibit the gene expression of signal transducer and activator of transcription factor 1（STAT 1 ） in human esophageal squamous cell carcinoma cell line Eca109 by RNAinterference and investigate its effect on the radiosensitivity and cell cycle of Eca109 cells. Methods Interference vector pSTAT 1 -shRNA for STAT 1 gene was designed and constructed. After being mixed with lentiviral packaging plasmids, the interference vectors were used to transfected 293T cells. Virus solution was collected to infect ECA109 cells. Real-time PCR and Western blot were used to measure the mRNA and protein expression levels of STAT 1 in Eca109 cells. Colony formation assay and flow cytometry were used to evaluate the radiosensitivity and cell cycle distribution of Eca109 cells.Results All Eca109 cells were divided into blank control group, transfection-positive group, and transfection-negative group. The transfection-positive group showed significantly lower mRNA and protein expression levels of STAT 1 than the other two groups. The values of D 0 , SF 2 , and D q of transfection-positive group were 2.03 Gy, 0.83, and 1.20 Gy, respectively, lower than those of blank control group （2.98 Gy, 0.88, and 1.39 Gy） and those of transfection-negative cells （3.02 Gy, 0.88, and 1.57 Gy）. At 12 h, 24 h, and 48 h after 4 Gy-irradiation, the transfection-positive group showed significantly higher percentage of G 0 ＋G 1 than the blank control group and transfection-negative group （34.13% vs 22.03% vs 22.27%, F=7.56, P=0.023;43.80% vs 28.40% vs 28.63%, F=10.01, P=0.012;53.20% vs 42.2% vs 41.83%, F=10.73, P=0.010） and significantly lower percentage of G 2 ＋M than the blank control group and transfection-negative group （14.33% vs 32.23% vs 32.23%, F=16.86, P=0.003;27.73% vs 43.53% vs 44.00%, F=26.62, P=0.001;14.23% vs 27.97% vs 27.93%, F=40.34, P=0.000）.Conclusions RNA interference of STAT 1 in Eca109 cells does not affect the proliferation ability of Eca109 cells, and it can increase the radiosensitivity of Eca109 cells probably by regulating cell cycle after irradiation.