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
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东南大学913/事业基金资助项目(9290001327)


Downregulation of hENT1 by RNAi enhances G0/G1 phase arrest of 5-FU to pancreatic cancer cells
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    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(600 -滋g/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(20 μmol/L)中24 h后的细胞周期-结果:测序显示重组质粒pSilence-hENT1 4.1-CMV neo中插入片段序列正确-RT-PCR结果显示pSilence-hENT1 Panc-1细胞hENT1 mRNA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-

    Abstract:

    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-hENT1 4.1-CMV neo was transfected into Panc-1 cells with Lipofectamin 2000. After that, the transfected cells(pSilence-hENT1 Panc-1) was selected with medium containing G418(600 -滋g/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 hENT1-mRNA expression by RNAi enhances G0/G1 phase arrest of 5-FU to pancreatic cancer cells.

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陈武强,刘胜利,徐建敏. 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[J].南京医科大学学报(自然科学版),2009,29(2):177-181210

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  • 收稿日期:2008-09-27
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