Chen Jian-yong, Luo Bin, Guo Xiao-bai, Wan Li-hui
Department of Gastroenterology, The Jiangxi Provincial People's Hospital, Nanchang 330006, China.
Zhonghua Zhong Liu Za Zhi. 2011 Feb;33(2):91-6.
The aim of this study was to construct a recombinant plasmid carrying fusion suicide gene CDglyTK and RNA interference eukaryotic expressing vector targeting to STAT3, and to investigate the effect of double suicide gene combined with RNAi targeting to STAT3 on HCT116 and HUVEC cells in vitro.
The CD and TK were cloned by polymerase chain reaction (PCR), and fusion gene CDglyTK was inserted into plasmid pEGFP after DNA sequence analysis, enzyme digestion and ligation. The recombinant plasmid was analyzed by PCR amplification and electrophoresis and enzyme digestion. DNA sequences containing small hairpin structure targeting to STAT3 were synthesized and inserted into the vector. The CDglyTK gene expressions in HCT116 and HUVEC cells were examined by reverse transcription-polymerase chain reaction (RT-PCR) after transfection of HCT116 and HUVEC cells. The inhibitory effect of RNA interference vector targeting to STAT3 was analyzed by RT-PCR and Western blot. The effects of 5-FC and GCV on HCT116 and HUVEC cells transfected with the recombinant plasmids were detected by MTT staining.
The results of restriction enzyme digestion and PCR amplification and electrophoresis showed that the recombinant pEGFP/CDglyTK was constructed correctly. The mRNA expression of gene CDglyTK was detected in HCT116 and HUVEC cells which transfected with the recombinant plasmid. The results of RT-PCR and Western blot showed that the RNA interference expression vector targeting to STAT3 effectively inhibited the expression of STAT3 in HCT116 cells. The results of MTT test showed that the inhibition ratio of group pEGFP/CDglyTK was (63.72 ± 0.64)%, significantly higher than that of control group (P < 0.05). The inhibition rate of group pEGFP/STAT3 siRNA was (47.02 ± 0.39)%, which was lower than that of group pEGFP/CDglyTK (P < 0.05), and higher than that of control group (P < 0.05). The inhibition rate of group pEGFP/CdglyTK + pEGFP/STAT3 siRNA was (85.10 ± 0.17)%, significantly higher than those of groups pEGFP/CDglyTK and group pEGFP/STAT3 siRNA (P < 0.05). Meanwhile, in HUVEC cells, the inhibition rate of group pEGFP/CDglyTK was (70.24 ± 0.33)%, significantly higher than that of the control group (P < 0.05). The inhibition rate of group pEGFP/STAT3 siRNA was (46.32 ± 0.15)%, significantly lower than that of group pEGFP/CdglyTK (P < 0.05), and higher than that of the control group (P < 0.05). The inhibition rate of group pEGFP/CdglyTK+pEGFP/STAT3 siRNA was (87.10 ± 0.24)%, significantly higher than those of groups pEGFP/CDglyTK and pEGFP/STAT3 siRNA(P < 0.05).
The recombinant plasmids pEGFP-CDglyTK and pEGFP/STAT3 siRNA have inhibitory effect on HCT116 and HUVEC cells. The killing effects of double suicide gene combined with RNAi targeting to STAT3 are much better than those of single gene therapy.
构建携带融合自杀基因CDglyTK的重组质粒及靶向信号转导与转录激活因子3（STAT3）的RNA干扰真核表达载体，探讨双自杀基因联合靶向STAT3的RNA干扰对人结肠癌细胞系HCT116和人脐静脉内皮细胞（HUVEC）的体外杀伤作用。
采用聚合酶链反应（PCR）克隆胞嘧啶脱氨酶（CD）和胸苷激酶（TK）基因，经DNA序列分析、酶切及连接后，将融合基因CDglyTK插入质粒pEGFP。对重组质粒进行PCR扩增、电泳及酶切鉴定。合成含靶向STAT3的小发夹结构的DNA序列并插入载体。将重组质粒转染HCT116和HUVEC细胞后，用逆转录-聚合酶链反应（RT-PCR）检测细胞中CDglyTK基因的表达。采用RT-PCR和蛋白质免疫印迹法（Western blot）分析靶向STAT3的RNA干扰载体的抑制作用。用噻唑蓝（MTT）染色法检测5-氟胞嘧啶（5-FC）和丙氧鸟苷（GCV）对转染重组质粒的HCT116和HUVEC细胞的作用。
酶切、PCR扩增及电泳结果显示重组质粒pEGFP/CDglyTK构建正确。转染重组质粒的HCT116和HUVEC细胞中可检测到CDglyTK基因的mRNA表达。RT-PCR和Western blot结果显示，靶向STAT3的RNA干扰表达载体可有效抑制HCT116细胞中STAT3的表达。MTT检测结果显示，pEGFP/CDglyTK组的抑制率为（63.72±0.64）%，显著高于对照组（P<0.05）。pEGFP/STAT3 siRNA组的抑制率为（47.02±0.39）%，低于pEGFP/CDglyTK组（P<0.05），高于对照组（P<0.05）。pEGFP/CdglyTK+pEGFP/STAT3 siRNA组的抑制率为（85.10±0.17）%，显著高于pEGFP/CDglyTK组和pEGFP/STAT3 siRNA组（P<0.05）。同时，在HUVEC细胞中，pEGFP/CDglyTK组的抑制率为（70.24±0.33）%，显著高于对照组（P<0.05）。pEGFP/STAT3 siRNA组的抑制率为（46.32±0.15）%，显著低于pEGFP/CdglyTK组（P<0.05），高于对照组（P<0.05）。pEGFP/CdglyTK+pEGFP/STAT3 siRNA组的抑制率为（87.10±0.24）%，显著高于pEGFP/CDglyTK组和pEGFP/STAT3 siRNA组（P<0.05）。
重组质粒pEGFP-CDglyTK和pEGFP/STAT3 siRNA对HCT116和HUVEC细胞有抑制作用。双自杀基因联合靶向STAT3的RNA干扰的杀伤效果优于单基因治疗。
