RNA干扰PDK-1对5-FU诱导结肠癌细胞凋亡的促进作用  

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

作　　者：谢贛丰 梁后杰[1] 童晶涛[1] 何金霞[1] 

机构地区：[1]第三军医大学西南医院肿瘤科,重庆400038

出　　处：《第三军医大学学报》2012年第1期9-12,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(30873015)~~

摘　　要：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。Objective To study the effect of pyruvate dehydrogenase 5-fluorouracil （5-FU）-induced apoptosis of colon cancer cells. Methods kinase isozyme 1 （ PDK-1 ） on Three interference sequences （interference group） and a negative control sequence of PDK-I shRNA （control group） were designed to con- struct 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 Mq^F assay and flow cytometry, respectively. Results Real-time PCR and Western blot- ting 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%. MrlT 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 ceils.

关 键 词：结肠肿瘤 丙酮酸脱氢酶激酶-1 细胞增殖 化学治疗 凋亡 

分 类 号：R394-33[医药卫生—医学遗传学] R730.23[医药卫生—基础医学]