胃癌相关基因GCRG213小干扰RNA表达载体的构建及其对胃癌细胞生长特性的影响

Construction of gastric cancer-related gene GCRG213 specific siRNA expression vector and its effect on MKN45 gastric cancer cells

  • 摘要: 目的 研究胃癌相关基因GCRG213 siRNA转染对胃癌细胞MKN45的影响。方法 1)设计两对针对GCRG213可转录为小干扰RNA(siRNA)的DNA片段,退火后插入小干扰RNA表达载体IMG-800。2)测序正确的重组子IMG-800-1,IMG-800-2和空载体转染MKN45细胞,采用RT-PCR及Western法比较GCRG213在mRNA和蛋白质水平上的表达差异。3)绘制细胞生长曲线,分析细胞的增殖状态,检测凋亡细胞。结果 1)干扰片段正确插入小干扰RNA表达载体IMG-800。2)重组子和空载体转染MKN45细胞。3)转染siRNA的MKN45细胞中其mRNA的表达下调44.9%和49.5%;其蛋白的表达下调55.3%和64.2%。4)转染siRNA的MKN45细胞生长增殖速度减慢,处于G0-G1期的细胞增加,G2/M期和/或S期的细胞比例减少,细胞凋亡率增加。结论 siRNA转染可抑制肿瘤细胞的生长和增殖,促进肿瘤细胞的凋亡。

     

    Abstract: Objective To investigate the effect of gene GCRG213 siRNA transfection on the growth of MKN45 gastric cancer cells. Methods Two pairs of DNA sequences containing small hairpin structure of GCRG213 were designed and synthesized. A complement form was obtained by annealing and inserted into the RNAi expression vector IMG 800. Recombinant plasmid and vector were transfected separately into MKN45 cells. Expression of GCRG213 was detected by semi-quantitative RT-PCR and Western blot. Growth curves for MKN45 cells were plotted. FACS was used to detect cell cycle and two Annexin V FITC/PI parameters were used to detect the effects of gene GCRG213 siRNA transfection on cell apoptosis. Results The two pairs of DNA sequences containing small hairpin structure of GCRG213 were successfully cloned into the siRNA expression vector IMG 800. The recombinant plasmid and vector were transfected separately into MKN45 cells. Transfecting the siRNA vector into MKN45 cells significantly decreased the expression of GCRG213 at mRNA and protein level. The growth of MKN45 cells was slower than that of vector-transfected cells. The proportion of cells in Gz/M and/or S period was decreased and cell apoptosis was increased. Conclusion SiRNA transduction can inhibit the growth and proliferation of MKN4 cells,and promoted cell apoptosis.

     

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