RNA干扰PDK-1对5-FU诱导结肠癌细胞凋亡的促进作用

目的探讨以丙酮酸脱氢酶激酶-1(pyruvate dehydrogenase kinase isozyme 1,PDK-1)为靶标的短发夹RNA(shRNA)与化疗药5-氟尿嘧啶(5-FU)联合应用对人结肠癌LS174T细胞的促凋亡作用。方法针对PDK-1序列设计3段干扰序列及1个阴性对照,分别构建pGenesil.1-PDK-1重组质粒表达载体及pGenesil.1-NC阴性对照载体,利用脂质体分别将质粒转染入LS174T,并用G418对细胞进行药物筛选,并通过Real-time PCR和Western blot检测有干扰序列的有效性。MTT实验检测5-FU对干扰组和对照组细胞生长、增殖的影响,流式细胞仪检测细胞凋亡率。结果 Real-time PCR和Western blot证实3个干扰组均抑制PDK-1表达,其中最大敲减效率超过70%。MTT实验结果显示,经5-FU同样处理48 h后,PDK-1干扰的LS174T细胞生长抑制率较对照组显著增高(P<0.05);流式细胞仪结果显示,干扰组细胞凋亡率较对照组增高(P<0.05)。结论 PDK-1 shRNA能特异性下调LS174T细胞中PDK-1的表达,PDK-1干扰联合5-FU可促进结肠癌细胞化疗诱导凋亡。

Promoting effect of RNA-interfered pyruvate dehydrogenase kinase isozyme-1 on 5-fluorouracil-induced apoptosis of colon cancer cells

Objective To study the effect of pyruvate dehydrogenase kinase isozyme 1(PDK-1) on 5-fluorouracil(5-FU)-induced apoptosis of colon cancer cells.Methods Three interference sequences(interference group) and a negative control sequence of PDK-1 shRNA(control group) were designed to construct pGenesil-1 recombinant plasmid expression vector and pGenesil.1 negative control(pGenesil.1-NC) plasmid vector,respectively.The plasmid was transfected into LS174T cells using liposome and drugs were screened with G418.Effect of RNA interference on 5-FU-induced apoptosis of colon cancer cells was detected by real-time PCR and Western blotting,respectively.Effect of 5-FU on growth and apoptosis of colon cancer cells was detected by MTT assay and flow cytometry,respectively.Results Real-time PCR and Western blotting showed that the 3 interference sequences of PDK-1 shRNA inhibited the expression of PDK-1 in LS174T cells,with a maximum inhibitory rate of over 70%.MTT assay demonstrated that the growth inhibiting rate of LS174T cells interfered with PDK-1 was significantly higher in interference group than in control group 48 h after 5-FU was used(P<0.05).Flow cytometry displayed that the apoptosis rate of colon cancer cells was significantly higher in interference group than in control group(P<0.05).Conclusion PDK-1 shRNA can down-regulate the expression of PDK-1 in LS174T cells and PDK-1 interference in combination with 5-FU can promote the apoptosis of colon cancer cells.