RNA干扰靶向抑制胃癌细胞血管内皮生长因子C表达的实验研究

Experimental research of the inhibition of vascular endothelial growth factor C expression in gastric cancer by targeting RNA interference

目的 构建针对血管内皮生长因子C（VEGF-C）的RNA干扰质粒表达载体pSIH1-H1-copGFP,并评价其转染胃癌细胞后对VEGF-C表达的抑制作用.方法 设计并合成针对VEGF-C基因mRNA序列的3条短发夹RNA及1条阴性对照序列,克隆到pSIH1-H1-copGFP载体,重组构建RNA干扰质粒,并将其转染胃癌细胞（SGC7901）,采用RT-PCR分析转染后VEGF-C基因的表达情况.结果 重组构建pSIH1-H1-copGFP载体经双酶切及插入片断序列分析,表明其成功插入设计位点,并且序列完全一致.3种siRNA质粒表达载体的转染效率均为60%～70%,对VEGF-C mRNA表达的抑制率分别为35.4%、33.8%和81.5%.结论 RNA干扰质粒表达载体介导对VEGF-C基因的RNA干扰可明显抑制胃癌细胞中VEGF-C的表达,可能成为抑制胃癌淋巴管生成的一种有效手段.

Objective To construct the plasmid expression vector pSIH1-H1-copGFP for RNA interference against vascular endothelial growth factor C（VEGF-C） and to evaluate its effect on the expression of VEGF-C mRNA in gastric cancer cells after transfection.Methods Three siRNAs of genome sequence of VEGF-C gene were retrieved from Gen Bank and one negative chain was used as control.Four siRNAs were cloned into plasmid pSIH1-H1-copGFP,which were then transfected into gastric cancer cells （SGC7901）.The expression of VEGF-C mRNA was analyzed by RT-PCR.Results The recombinant plasmid of pSIH1-H1-copGFP specific for VEGF-C was confirmed by gene sequencing analysis.The target sequence obtained was completely consistent with the design.Transfection efficiency of the three siRNAs ranged from 60% to 70%.After transfection,the expression of VEGF-C mRNA in SGC7901 cells was significantly inhibited.Inhibition rates of VEGF-C mRNA expression were 35.4%,33.8% and 81.5% in the three siRNA plasmid vectors,respectively.Conclusion The siRNA expression plasmid vector against VEGF-C mRNA is successfully constructed,and RNAi may be a useful technique to inhibit the lymphangiogenesis of gastric cancer.