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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 design an interference plasmids targeting MTDH gene by RNAi technique and observe the interference effect of liposome-transfected esophageal cancer cells EC9706 to evaluate the effect of MTDH gene on cell proliferation and cell cycle. Methods Three pairs of interference sequences targeting MTDH gene were designed and named MTDH-shRNA-1, MTDH-shRNA-2 and MTDH-shRNA-3, respectively. Another control-shRNA sequence independent of the MTDH gene was synthesized. The synthesized single-stranded DNA was denatured and annealed to form a double-stranded DNA. The double-stranded DNA was ligated to the pRNA-U6.1 / Neo linear plasmid digested with BamHI and HindIII. The experiment set up three groups: negative control group (normal growth EC9706 cells without any treatment), Control-shRNA group (transfected with random sequence Control-shRNA), MTDH-shRNA group (transfected MTDH-shRNA) Ability, cell cycle distribution. Results After transfection of EC9706 cells with MTDH-shRNA-1, MTDH-shRNA-2, MTDH-shRNA-3 and Control-shRNA, the percentages of green fluorescent cells accounted for> 80% MTDH-shRNA-1, MTDH-shRNA-2, and MTDH-shRNA-3 could significantly down-regulate the expression of MTDH mRNA in each group compared with the negative control group, the difference was statistically significant (P <0.05). Especially, the expression of MTDH mRNA in shRNA-1 group was significantly lower than the other two groups. Select MTDH-shRNA-1 (MTDH-shRNA) for cell function test. The proliferation rate of MTDH-shRNA group was significantly lower than MTDH-Control group and negative control group, the difference was statistically significant (P <0.05), MTDH-shRNA group and negative control group and MTDH-Control group, G0 / Accounting for an increase, while the proportion of S phase cells decreased, the difference was statistically significant (P <0.05). Conclusion The MTDH shRNA plasmid was constructed successfully. MTDHshRNA-1 was the best one. MTDH-shRNA inhibited cell proliferation and blocked the cell cycle of EC9706 cells in the S phase of cell cycle. Plasmid construction for the future MTDH in esophageal cancer and other research has laid the foundation.