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的影响

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

目的探讨胃癌相关基因GCRG213小干扰RNA（siRNA）转染对胃癌细胞MKN45的影响。方法设计两对针对GCRG213可转录为siRNA的DNA片段，退火后插入siRNA表达载体IMG-800。测序正确的重组子IMG-800-1（含干扰片段1）、IMG-800-2（含干扰片段2）和空载体经脂质体转染人胃癌细胞系MKN45细胞，G418筛选获得稳定转染的细胞株。采用半定量RT-PCR及Western免疫印迹法，比较转染不同质粒的MKN45细胞中GCRG213在mRNA和蛋白质水平上的表达差异。选取稳定转染不同质粒的MKN45细胞，采用细胞计数法绘制细胞生长曲线，流式细胞仪分析细胞的增殖状态，AnnexinV FTTC／PI双标记法检测凋亡细胞，平板克隆形成实验、裸鼠移植瘤实验分析转染细胞成瘤性。结果经测序证实，干扰片段退火后正确插入小干扰RNA表达载体IMG-800，组成重组子IMG-800-1和IMG-800-2。重组子IMG-800-1、IMG-800-2和空载体经脂质体转染人胃癌细胞系MKN45细胞，G418筛选获得稳定转染的细胞株。与对应的空载体比较，RT-PCR结果显示，转染siRNA的MKN45细胞中，mRNA的表达分别下调44．9％和49．5％；Western印迹法结果显示，转染siRNA的MKN45细胞中，蛋白的表达分别下调55．3％和64．2％。与转染空载体的细胞相比，转染siRNA的MKN45细胞生长增殖速度明显减慢，细胞周期表现为处于G0—G1期的细胞有所增加，G2／M期和（或）S期的细胞比例减少，细胞凋亡率增加，细胞克隆形成数量减少，裸鼠体内成瘤性降低。结论胃癌相关基因GCRG213 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，抑制肿瘤细胞的成瘤性。

Objective To investigate the effect of gene GCRG213 siRNA transfection into gastric cancer cell line MKN45 cells. Methods Two pairs of DNA sequences containing small hairpin structure to GCRG213 were designed and synthesized. The complement form was obtained by annealing and inserted into RNAi expression vector IMG-800. They are IMG-800-1 and IMG-800-2 correspondingly. The recombinant plasmid LMG-800-1, IMG-800-2 and the vector IMG-800 were separately transfected into MKN45 cells conducted by lipofectamineTM 2000. After G418 selecting, the cells were transfected steadily. Expression of GCRG213 was detected by semi-quantitative RT-PCR and Western Blot. The growth graph of six steady transfected cell cultures was protracted by cell counting. FACS was used to detect the cell cycle, and Annexin V FITC/PI double labeling were used to detect the effects on cell apoptosis in the above-mentioned cells. The clone formation rate in plate and in nude mice was tested to investigate the tumorigenic characteristics of the six steadily transfected cells in vitro and vivo. Results Through sequencing, two pairs of DNA sequences containing small hairpin structure to GCRG213 were proved to be successfully cloned into siRNA expression vector IMG-800, correspondingly called IMG-800-1 and IMG-800-2. The recombinant plasmid IMG-800-1, IMG-800-2 and vector IMG-800 were transfected separately into MKN45 cells conducted by lipofectamine^TM 2000. After G418 selecting, the cells were transfected steadily. Transfecting the siRNA vector （IMG-800-1, IMG-800-2 ） into the MKN45 cells significantly decreased the expression of GCRG213, at both mRNA and protein levels. The growth graph showed that the growth of IMG-800-1 and IMG-800-2 transfected cells were slower than that of vector transfected cells. The proportion of cells in G2/M and/or S phase decreased in the cells transfected with IMG-800-1 and IMG-800-2 and cell apoptosis increased. The average clone formation rate in vitro decreased in the cells transfected with IMG-800-1 and IMG-800-2, compared with those transfected with vector. In vivo, the time of tumor formation of IMG-800-1 and IMG - 800-2 transducted cells in nude mice was prolonged and the tumor size was smaller. Conclusion GCRG213 SiRNA transfection may induce inhibition of growth and proliferation of tumor cells, promote cell apoptosis, and inhibit the tumorigenicity in vitro and vivo.