RNAi下调hENT_1可增强5-氟尿嘧啶对胰腺癌细胞的毒性

Down-regulation of hENT_1 by RNAi increasing the cytotoxicity of 5-fluorouracil to pancreatic cancer cells

目的:运用RNAi技术下调平衡型核苷转运蛋白1(hENT1)的表达,观察hENT1下调后5-氟尿嘧啶(5-FU)对胰腺癌Panc-1细胞的毒性改变情况。方法:设计并合成能表达特异性针对hENT1的shRNA(smallhairpinRNA)的DNA序列,构建pSilence-hENT14.1-CMVneo质粒。用脂质体法将pSilence-hENT14.1-CMVneo质粒转染胰腺癌Panc-1细胞,并以含G418(600μg/mL)的培养液筛选阳性转染细胞(pSilence-hENT14.1-CMVneoPanc-1)。RT-PCR检测下调pSilence-hENT14.1-CMVneoPanc-1细胞hENT1mRNA表达的效果及稳定性。MTT法检测细胞在含5-FU(0～1.56×106nmol/L)的培养液中48h后的生存率,并计算IC50。结果:测序显示重组质粒pSilence-hENT14.1-CMVneo中插入片段序列正确。RT-PCR结果显示,pSilence-hENT14.1-CMVneoPanc-1细胞hENT1mRNA表达水平下调,并在两个月内(共传代15次)保持相对稳定。MTT结果显示,pSilence-hENT14.1-CMVneoPanc-1细胞组氟尿嘧啶的IC50显著下降,为Panc-1细胞组IC50的51.52%(P<0.01)。结论:用RNAi方法下调hENT1mRNA表达可增强5-FU对胰腺癌细胞的毒性。

Aim：To investigate whether down-regulation of human equilibrative nucleoside transporter 1 （hENT1） mRNA expression by RNAi would increase the cytotoxicity of 5-fluorouracil （5-FU） to Panc-1 cell line. Methods：A DNA fragment which expresses shRNA （small hairpin RNA） specifically hENT1 was designed and synthesized. This DNA fragment was inserted into pSilence 4.1-CMV neo plasmid. Then, the recombined plasmid （pSilence-hENT1 4.1-CMV neo） was transfected into Panc-1 cells with Lipofectamin 2000. Moreover, the transfected cells （pSilertce-hENTl 4.1- CMV neo Panc-1） selected with medium containing G418 （600 μg/mL）. hENT1-mRNA expression was detected by RT-PCR in order to determine whether it was efficiently and constantly down-regulated by RNAi. The cells were cultured in the media with the addition of a serial concentrations of 5-FU from 0 to 1.56 ×10^6 nmol/L for 48 h. Then the cell survival rate after 48 h was measured by MTI＇, and the IC50 of 5-FU was calculated. Results： pSilence-hENT1 4.1- CMV neo sequencing showed that the designed DNA fragment was successfully inserted into pSilence 4.1-CMV neo plasmid. The down-regulation of hENT1 -mRNA expression existed and it was kept stable for 2 months （ 15 passages） in pSilence-hENTl 4.1-CMV neo Panc-1 cell. However, the IC50 of 5-FU in pSilence-hENTl 4.1-CMV neo Panc-1 group was decreased to 51.52 % that of the control （P 〈 0.01）. Conclusion：The down-regulation of hENTl-mRNA expres- sion with RNAi increases the cytotoxicity of 5-FU to pancreatic cancer cells.