慢病毒介导shRNA抑制垂体腺瘤PTTG基因表达及对细胞增殖和侵袭能力的影响  

Inhibition of PTTG Expression and Suppression of Tumor Proliferation and Invasion in GH3 and At T20 Pituitary Adenomas Cells by Lentiviral Vector-Mediated sh RNA Targeting PTTG

作　　者：沈云龙[1] 安泰学 罗巧灵[1] 李和珍[1] 杨勇[1] 王雪娟[1] 刘丽红[1] 王芳[3] 

机构地区：[1]南方医科大学第五附属医院脑外科,广东广州510000 [2]南方医科大学第五附属医院检验医学系,广东广州510000 [3]中山大学附属肿瘤防治中心分子诊断科,广东广州510060

出　　处：《中山大学学报（医学科学版）》2016年第4期522-529,共8页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广东省科技计划项目(2013B021800050,2014A020212727);广州市科技和信息化局应用基础研究项目(2013J4100013)

摘　　要：【目的】观察慢病毒介导的sh RNA对GH3和At T20型垂体腺瘤中垂体肿瘤转化基因(PTTG)表达的抑制作用,以及对肿瘤细胞增殖和侵袭能力的影响及其机制探讨。【方法】筛选最佳抑制效果的干扰序列构建PTTG基因sh RNA的慢病毒表达载体,转染GH3和At T20型垂体腺瘤细胞。流式细胞仪分析细胞转染效率,RT-PCR和Western blot检测细胞PTTG基因m RNA和蛋白的表达水平,MTT和Ed U法分别检测靶向PTTG的sh RNA对肿瘤细胞增殖生长的抑制作用,流式细胞仪检测细胞周期变化。Transwell小室检测细胞侵袭能力的变化,基因芯片筛查干扰后的基因表达谱差异并进行生物信息学分析。【结果】细胞转染表达PTTG基因sh RNA的慢病毒载体后,能显著抑制肿瘤细胞PTTGm RNA和蛋白的表达,与对照组相比有统计学差异(P<0.05),细胞的增殖能力受到明显抑制,且增殖抑制效应具有时间依赖性;细胞生长显著变慢,G0/G1期细胞比例显著增高,克隆形成能力下降,细胞侵袭能力也显著减弱,均与对照组相比有统计学差异(P<0.05)。PI3K等信号通路的基因表达也发生显著变化。【结论】慢病毒介导sh RNA沉默PTTG基因表达可能是通过对PI3K信号通路的调控,抑制了GH3和At T20垂体腺瘤细胞的生长,抑制肿瘤细胞的体外增殖和侵袭能力。[Objective] To observe and explore the suppression of lentiviral shRNA on PTTG in GH3 and AtT20 pituitary adenomas cells and its effect on tumor cells＇ proliferation and invasion. [ Methods] A lentivirus shRNA expressing vector targeting PTTG gene was constructed and packaged.The rate of GH3 and ART20 pituitary adenomas cells infected lentivirus shRNA vector was analyzed by fluorescence activated cell sorter （FACS）. Expression level of PTFG mRNA and protein was analyzed by RT-PCR and Western blot. Cell proliferation was performed withMTT. Cell cycle was determined by flow cytometry. Transwell assay was used to determine the invasion ability of tumor cells. Affymetrix GeneChipprimeview was used. In microarray process to determine differences of gene expression profile. [Resuhs]Lentiviral-mediated shRNA efficiently reduced PTTG expression. PTTG knockdown impaired cell proliferation, colony formation andinvasion ability,and induced cell cycle arrest in GH3 and AtT20 cells. [Conclusion] PTFG gene silencing by shRNA mediated with lentiviral vector could significantly inhibit GH3 and AtT20 cells growth. It also can inhibit the proliferation and invasion of these pituitary adenomas cells. Microarray analysis revealed that multiple cancer-associated pathways and oncogenes were regulated by PTTG.

关 键 词：短发夹RNA 垂体腺瘤 垂体肿瘤转化基因 基因治疗 

分 类 号：R739.41[医药卫生—肿瘤]