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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(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 effect of 5-fluorouracil (5-FU) on the cell cycle arrest of pancreatic cancer Panc-1 cells by down-regulating the expression of hENT1 by RNAi technology. Methods: The recombinant plasmid pSilence-hENT1 4.1-CMV neo, which can express specific shRNA against hENT1, was designed and constructed. The recombinant plasmid was transfected into Panc-1 cells with Lipofectamin 2000 and transfected into Panc-1 cells with G418 ) Were screened for positive transfected cells (pSilence-hENT1 Panc-1). The effect and stability of hENT1 mRNA expression down-regulated by RT-PCR. Flow cytometry was used to detect the cell cycle of Panc-1 cells in the same concentration of 5-FU (20μmol / L) before and after hENT1 down-regulation. Results: Sequencing showed that the insert sequence of recombinant plasmid pSilence-hENT1 4.1-CMVneo was correct. RT-PCR results showed that the expression of hENT1 mRNA in pSilence-hENT1 Panc-1 cells was down-regulated and remained relatively stable within 2 months (15 passages in total). The results of cell cycle assay showed that the percentage of G0 / G1 phase in hENT1-down-regulated group was 1.20 times (P <0.01) and the percentage of S phase in untreated group under the same concentration of 5-FU was 66.76 % (P <0.01). CONCLUSION: Inhibition of hENT1 expression enhances the arrest of G0 / G1 phase of pancreatic cancer cells by 5-FU, which can enhance its antitumor effect.