MTDH靶向RNA干扰质粒构建及其对食管癌细胞EC9706的影响

The study on construction and screening of RNA interference plasmid of MTDH and its impact on Esophageal cancer cells EC9706

目的应用RNA干扰技术设计构建针对MTDH基因的干扰质粒,观察脂质体转染食管癌细胞EC9706的干扰效果,评价MTDH基因对细胞增殖及细胞周期的影响。方法设计针对MTDH基因的3对干扰序列,分别命名为MTDH-shRNA-1、MTDH-shRNA-2、MTDH-shRNA-3。另设计与MTDH基因无关序列的Control-shRNA,合成的单链DNA经变性、退火形成双链DNA与经过BamHI和HindⅢ双酶切后的pRNA-U6.1/Neo线性质粒连接。实验设立3组:阴性对照组(正常生长的EC9706细胞,不做任何处理)、Control-shRNA组(转染随机序列Control-shRNA)、MTDH-shRNA组(转染MTDH-shRNA),检测细胞增殖能力、细胞周期分布。结果将MTDH-shRNA-1、MTDH-shRNA-2、MTDH-shRNA-3及Control-shRNA转染EC9706细胞后,48h时倒置显微镜下观察可见绿色荧光细胞所占细胞的百分比>80%,干扰载体能有效表达,转染各组与阴性对照组比较,MTDH-shRNA-1、MTDH-shRNA-2、MTDH-shRNA-3组对MTDH mRNA的表达都有明显的下调,差异有统计学意义(P<0.05),尤其是shRNA-1组的MTDH mRNA的表达明显低于其它2组。选取MTDH-shRNA-1(MTDH-shRNA)进行细胞功能的试验。MTDH-shRNA组细胞增殖速度明显低于MTDH-Control组及阴性对照组,差异有统计学意义(P<0.05),MTDH-shRNA组与阴性对照组及MTDH-Control组比较,G0/G1期所占比例增加,而S期细胞所占比例减少,差异有统计学意义(P<0.05)。结论成功构建MTDH干扰质粒,MTDHshRNA-1干扰效果最好,MTDH-shRNA抑制细胞增殖,使EC9706细胞的细胞周期阻滞在细胞分裂周期的S期。质粒的构建为今后MTDH在食管癌等肿瘤的研究方面奠定了基础。

Objective To investigate the effect of designed and synthesized the small hairpinRNA（shRNA）in Esophageal cancer cells EC9706.Methods RNA interference plasmid for MTDH gene was desiged and construacted.The MTDH-shRNAs sequences were named as：MTDH-shRNA-1,MTDH-shRNA-2and MTDH-shRNA-3.We also designed unrelated sequence of Control-shRNA.Synthesized single-stranded DNA was denatured and annealed to form a double-stranded DNA and connected with linearized plasmid pRNA-U6.1/Neo by Bam HI and Hind Ⅲ.We established 3groups：negative control group（normal growth of EC9706 cells,without any treatment）,Control-shRNA group（transfected with random sequence Control-shRNA）,MTDH-shRNA（transfected MTDH-shRNA）,cell proliferation and cell cycle distribution were detected.Results MTDH-shRNA-1,MTDH-shRNA-2,MTDH-shRNA-3and ControlshRNA transfected EC9706 cells were observed under an inverted microscope,the percentage of green fluorescent cells was80%interference.Compared with negative control group MTDH mRNA expression of shRNA-1,shRNA-2,shRNA-3was significantly down-regulated in each group,the difference was statistically significant（P 〈0.05）.MTDH mRNA expression of shRNA-1group was significantly lower than the other two groups.Therefore,in present study,MTDH-shRNA-1（hereinafter referred to MTDHshRNA）was selected for evaluation the cell function.The growth rate of MTDH-shRNA group was significantly lower than MTDH-Control group and negative control group,with significant differences（P 〈0.05）,MTDH-shRNA transfected with the negative control group and MTDH-Control group,the proportion of G0/G1 phase was lower and S-phase was higher in MTDH-shRNA group compared with MTDHControl group and negative control group（P 〈0.05）.Conclusion MTDH interference plasmid was successfully constructed,MTDH-shRNA-1interference was most efficient,MTDH-shRNA inhibited cell proliferation,cell cycle arrests in S phase of EC9706 cells.Plasmid was usefull for future research of MTDH in esophageal cancer and other tumors of anti-tumor therapy foundation