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pcDNA3.1-KISS-1真核表达载体的构建及其对EC1细胞转移的抑制作用

Construction of Eukaryotic Expression Plasmid pcDNA3.1-KISS-1 and Its Metastasis Inhibiting Effect to EC1 Cells

  • 摘要: 目的 克隆人食管组织KISS-1基因及构建真核表达载体pcDNA3.1-KISS-1,并观察其在食管癌EC1细胞中的表达及对EC1细胞侵袭及运动能力的影响。 方法 从人正常食管组织中提取总RNA,设计特异性引物,通过逆转录聚合酶联链反应(RT-PCR)扩增KISS-1基因cDNA序列,并将其克隆到真核表达载体pcDNA3.1中,构建真核表达质粒pcDNA3.1-KISS-1,酶切鉴定及测序分析后用Lipofectamine脂质体将其转染到食管癌细胞,Western blot法检测其蛋白在EC1细胞中的表达情况;应用细胞运动实验及重建基底膜侵袭实验分析KISS-1表达对EC1细胞运动和侵袭能力的影响。 结果 成功克隆了人KISS-1基因全长编码序列,构建了重组真核表达载体pcDNA3.1-KISS-1,脂质体转染后KISS-1基因在EC1细胞中成功表达;转基因组细胞的运动能力(174.6±14.24)和体外穿越重建基底膜的能力(90.44±12.83)明显低于转空质粒组(222.6±30.08,110.22±15.87)和对照组EC1细胞(234.40±14.72,124.78±18.14)(P均<0.05)。 结论 重组pcDNA3.1-KISS-1真核表达质粒构建成功,并且KISS-1对食管癌细胞的侵袭和运动能力具有抑制作用。

     

    Abstract: Objective To clone human esophageal tissue KISS-1 gene and construct its eukaryotic expression vector pcDNA3.1-KISS-1.To detect its expression in EC1 cells and observe the influence of transfection KISS-1 gene on the invasion and migration ability of EC1 cells. Methods Total RNA was extracted from human esophageal tissue.KISS-1 cDNA was isolated by using RT-PCR,and cloned into the eukaryotic expression vector pcDNA3.1.The eukaryotic expression plasmid pcDNA3.1-KISS-1 was verified by sequencing and enzyme digestion analysis.Using liposome-mediated transfection technique,the eukaryotic expression vector pcDNA3.1-KISS-1 was transfected into EC1 cells.The expression of protein in EC1 cells was detected by Western blot.The effect of KISS-1 expression on the invasion and migration of EC1 cells was investigated by Boyden-chamber system. Results pcDNA3.1-KISS-1 recombinant plasmid was successfully constructed and it had a higher level expressed in EC1 cells.Matrigel invasion and migration assay showed that the invasion and migration of cells transfected with KISS-1 were less than those in cells transfected with pcDNA3.1 and control cells(P<0.05). Conclusion The expression of KISS-1 could inhibit EC1 cells invasion and migration in vitro.

     

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