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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 investigate the effect of short hairpin RNA (shRNA) targeted to pyruvate dehydrogenase kinase 1 (PDK-1) and 5-fluorouracil (5-FU) combined with chemotherapeutic drug on human colon cancer LS174T Apoptosis of cells. Methods A three-segment interference sequence and a negative control were designed according to PDK-1 sequence. The recombinant plasmid pGenesil.1-PDK-1 and the pGenesil.1-NC negative control vector were constructed respectively. The plasmids were transfected into LS174T, and the cells were screened by G418, and the effectiveness of the interference sequences was tested by Real-time PCR and Western blot. MTT assay was used to detect the effect of 5-FU on cell growth and proliferation in the interference group and the control group, and the apoptosis rate was detected by flow cytometry. Results Both Real-time PCR and Western blot confirmed that PDK-1 expression was inhibited by the three interference groups, and the maximal knockdown efficiency was over 70%. MTT assay showed that the growth inhibition ratio of LS174T cells treated with 5-FU for 48 h was significantly higher than that of the control group (P <0.05). The results of flow cytometry showed that the apoptosis rate of LS174T cells was significantly higher than that of the control group The control group increased (P <0.05). Conclusion PDK-1 shRNA can specifically down-regulate the expression of PDK-1 in LS174T cells, PDK-1 interference combined with 5-FU can promote chemotherapy-induced apoptosis of colon cancer cells.