RNAi下调hENT1可增强5-氟尿嘧啶对胰腺癌细胞G0／G1期的阻滞作用

Downregulation of hENT_1 by RNAi enhances G0/G1 phase arrest of 5-FU 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细胞周期阻滞作用的改变情况。方法:设计并构建能表达特异性针对hENT1的shRNA的重组质粒pSilence-hENT1 4.1-CMV neo,用脂质体Lipofectamin 2000将重组质粒转染胰腺癌Panc-1细胞,并以含G418(600μg/ml)的培养液筛选阳性转染细胞(pSilence-hENT1 Panc-1)。RT-PCR检测下调hENT1 mRNA表达的效果及稳定性。用流式细胞仪检测hENT1下调前后Panc-1细胞在相同浓度5-FU(20μmol/L)中24h后的细胞周期。结果:测序显示重组质粒pSilence-hENT1 4.1-CMVneo中插入片段序列正确。RT-PCR结果显示pSilence-hENT1 Panc-1细胞hENT1mRNA表达水平下调,并在2个月内(共传代15次)保持相对稳定。细胞周期检测结果显示在相同浓度5-FU的作用下,hENT1下调组胰腺癌细胞的G0/G1期百分比为未下调组的1.20倍(P<0.01),S期百分比则是未下调组的66.76%(P<0.01)。结论:抑制hENT1的表达可以增强5-FU对胰腺癌细胞的G0/G1期阻滞作用,从而可提高其抗癌效果。

Objective：To investigate the alteration of cell cycle arrest of 5-FU to Panc-1 cell line after downregulation of hENT1 （human equilibrative nucleoside transporter 1） mRNA expression by RNAi. Methods：The plasmid （pSilence-hENT1 4.1-CMV neo） which expresses shRNA（small hairpin RNA） specifically for human equilibrative nucleoside transporter 1 （hENT1） was designed and recombined. Then, pSilence-hENTl 4.1-CMV neo was transfected into Panc-1 ceils with Lipofectamin 2000. After that, the transfected cells （pSilence-hENT1 Panc-1 ） was selected with medium containing G418 （600 μg/ml）, hENT1-mRNA expression was detected by RT-PCR to determine whether it was downregulated efficiently and stably by RNAi. Cells were cultured in the medium with 20 μmoL/L of 5-FU 24 h. Then the cell cycle was detected by flow cytometry （FCM）. 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 downregulation of hENT1-mRNA expression was found and it had maintained for 2 months （15 passages） in pSilence-hENT1 Panc-1 cell. The percentage of G0/G1 phase cells in pSilence-hENT1 Panc-1 group was increased to 120%（P 〈 0.01） of the Panc-1 group,and the percentage of S phase cells was reduced to 66.76%（P 〈 0.01） compared with Panc-1 group. Conclusion：The downregulation of hENTrmRNA expression by RNAi enhances G0/G1 phase arrest of 5-FU to pancreatic cancer cells.