反义PTTG真核表达载体对人卵巢癌细胞SK-OV-3恶性表型的抑制作用

背景与目的:垂体肿瘤转化基因(pituitarytumortransforminggene,PTTG)是一个新的原癌基因,具有多种促肿瘤生长与转移的作用。目前研究多集中于PTTG在各种肿瘤组织中的表达及其相关的调控机制,而针对其反义阻断可能性的报道较少。本研究中,我们通过构建携带能够表达全长反义PTTGmRNA的真核表达载体,观察其对人卵巢癌细胞株SK-OV-3的反义阻断效应。方法:利用含酶切位点的PCR引物克隆全长PTTG,反向插入真核表达载体pcDNA3.1;用脂质体将重组载体转染SK-OV-3细胞,G418筛选阳性克隆后,Westernblot检测PTTG蛋白和碱性成纤维细胞生长因子(basicfibroblastgrowthfactor,bFGF)表达的改变,MTT法检测细胞增殖情况,并利用软琼脂糖集落形成实验观察细胞生物学性状的变化。结果:筛选出稳定表达重组pcDNA3.1-PTTGas的SK-OV-3细胞;转染细胞组较未转染细胞组的PTTG、bFGF表达分别减少了61.5%和52.3%;转染细胞增殖明显增强;转染细胞在软琼脂中的集落形成数(2.4±0.8)明显低于未转染组和转染空白质粒对照组(23.3±5.7和21.5±7.9)(P<0.01)。结论:反义PTTG真核表达载体作为一种新的工具,为针对PTTG的肿瘤基因治疗提供了可能性。

Construction of PTTGas antisense expression vector and its inhibitory effects on human ovarian carcinoma cell line SK-OV-3

BACKGROUND & OBJECTIVE: Pituitary tumor transforming gene (PTTG) is a new proto-oncogene and shows multiple actions of promoting tumorigenesis and metastasis. Most researches mainly focus on the problems of PTTG expression in different tumor tissues and its relative regulatory mechanisms, but no research of exploring the possibility of antisense blocking of PTTG gene has been reported by now. In present study, the authors constructed eukaryotic expression vector expressing full-length anti-sense PTTG mRNA and observed its blocking effect on the potential invasion of human ovarian carcinoma cell line SK-OV-3. METHODS: PCR primers containing designed enzyme cut sites were used for cloning full-length PTTG gene fragment, and the resulting PCR product was inserted into the eukaryotic vector pcDNA3.1 in the antisense direction. The recombinant vector was then transfected into SK-OV-3 by lipofectamine. The positive cell clone was screened by G418,PTTG,and bFGF at protein level expression were detected by Western blot analysis. The changes of cell proliferation were analyzed by MTT method. The biological behavior change of transfection positive cells was observed by colony formation in soft agar assay. RESULTS: SK-OV-3 clones stably expressing full-length recombinant pcDNA3.1- PTTGas were obtained.The expression of PTTG and bFGF proteins in transfected cells were decreased by 61.5% and 52.3% respectively as compared with non-transfected ones. The cell proliferation was accelerated in transfected cells. The number of colony formation is reduced significantly in transfected cells (2.4+/-0.8) as compared with non-transfected and empty vector transfected cells (23.3+/-5.7 and 21.5+/-7.9, respectively, P< 0.01). CONCLUSION: The recombinant vector pcDNA3.1-PTTGas is a novel tool and brings us a new possibility of anti-sense gene therapy targeted at PTTG in human carcinoma.