RNA干扰53BP1基因表达对食管癌放射敏感性的影响

Effect of RNAi against 53BP1 gene on radiosensitivity of human esophageal carcinoma cells

背景与目的:细胞周期检测点激酶53BP1在细胞周期调控方面发挥着重要的作用,对体细胞的正常生长没有明显的调节作用,但在DNA损伤发生后被激活,引起细胞周期阻滞。本研究利用RNA干扰技术降低食管癌细胞ECA109细胞53BP1基因表达,观察其对放射线照射后细胞周期和放射敏感性的影响。方法:针对53BP1 mRNA序列,设计合成3对有效的干扰序列(siRNAl、siRNA2和siRNA3)和阴性对照序列,与载体pSIH1-H1-copGFP连接形成重组质粒,瞬时转染细胞,实时PCR(real-time PCR)和Western blot检测53BP1的表达,筛选有效干扰序列,与慢病毒包装质粒混合物共同转染293T细胞,收集病毒液,感染ECA109细胞,获得稳定转染细胞系,命名为ECA109/B,转染空载体组命名为ECA109/N。采用real-time PCR和Western blot测定53BP1在mRNA水平和蛋白水平的表达。采用MTT、流式细胞术和克隆形成实验检测RNA干扰53BP1基因对ECA109细胞放射敏感性的影响。结果:成功构建p53BP1-shRNA质粒,3对干扰序列对人53BP1基因的表达在mRNA和蛋白水平显著低于阴性对照组和空白对照组,尤以siRNA1组效果最明显,取干扰效果最好的siRNA1,利用慢病毒包装系统,共同转染293T细胞,收集病毒液,感染ECA109细胞,荧光显微镜下挑取阳性克隆,扩大培养,获得稳定转染细胞株ECA109/B,53BP1基因在mRNA和蛋白表达水平亦低于对照组;MTT结果表明53BP1低表达对细胞的增殖无明显影响,用流式细胞术分析显示5 Gy射线照射后,ECA109/B的G2/M期比例明显低于阴性对照组和空白对照组;克隆形成实验的结果显示ECA109、ECA109/N、ECA109/B细胞的D0值分别为3.06、2.90和2.07 Gy;SF2值分别为0.91、0.89和0.79;Dq值分别为1.59、1.47和1.21,可见ECA109/N、ECA109放射敏感性未见明显差异,而ECA109/B细胞的放射敏感性高于ECA109/N、ECA109。结论:RNA干扰技术可以有效地抑制食管癌细胞ECA109中53BP1基因的表达,从而增强ECA109细胞的放射敏感性。

Background and purpose： Cell cycle checkpoint kinases p53-binding protein 1 （53BP1） plays an important role in regulating cell cycle distribution. 53BP1 has no effect on the growth of somatical cells. When irradiation induces DNA damage, 53BP1 is activated immediately, and arrest of cell cycle is induced to enhance repair for DNA lesions. The study explored the influence of 53BP1 gene expression by RNAi on the radiosensitivity of human esophageal carcinoma cell line ECA109. Methods： Three pairs of siRNA based on the sequences of the 53BP1 mRNA were synthesized （siRNA1, siRNA2, and siRNA3）, and a negative one was synthesized to be used as control. 53BP 1 -siRNA positive recombinant plasmids （pSIH 1 -H 1 -siRNA1, pSIH 1 -H 1 -siRNA2, pSIH 1 -H 1 -siRNA3） were thus constructed and then transfected into the cultured ECA109 cells. Inhibitory effects of siRNA on 53BP1 mRNA and protein expression were detected by real-time PCR and Western blot respectively in instantaneously transfected cells. The siRNA with the best inhibitory effect or empty vector was co-transfected along with pPACKH1-LentivectorPackaging system into 293T to package lentivirus particles, 48 hours after transfection, stable integrants with specific or control lentiviral vectors, selected by using copGFP reporter gene, were defined as ECA109/B （pSIH1-HI-siRNA1） and ECA109/N （pSIH1-H 1-negtive）, respectively. The effect of siRNA was identified by real-time RT-PCR and Western blot. The effect of knockdown of 53BP1 on proliferation ability of Ecal09 cell was observed by MTT. The sensitivity of ECA109 cells to radiotherapy was detected by flow cytometry （FCM） and clone formation assay, so as to elucidate whether knockdown of 53BP1 can increase the sensitivity to radiotherapy. Results： Eukaryotic expression plasmid expressing siRNA targeting 53BP1 gene was constructed successfully. The results of real-time PCR and Western blot showed in instantaneously transfected cells, 53BP1 mRNA and protein expression were inhibited. The mRNA and protein expression level of 53BP1 in group transfected with pSIH1-HI-siRNA1 were both significantly lower than the other groups. The results of FCM showed the degree of retardance of G：/M stage was more serious in ECA109/B cells than that in ECA109 and ECA109/N cells. Percent of S stage and apoptosis were not influenced in this report. Do value and SF2 value were 3.06 Gy and 0.91 in ECA109 cells. Do values were 2.90, 2.07 Gy and SF2 values were 0.89, 0.79 respectively with clonegenetic assay in ECA109/N, ECA109/B cells. Conclusion： RNAi could inhibit 53BP1 gene expression and enhance radiosensitivity of ECA109 cell.