RNAi技术干扰人喉癌Hep-2细胞PTTG1基因的初步研究

目的:探索对人PTTG1基因mRNA序列有较高干扰效率的位点,构建对PTTG1基因的表达有较高抑制效率的重组质粒。方法:筛选、合成1对互补DNA单链,退火为双链后与双酶切后的质粒载体连接,构建了表达短发夹RNA的1种重组质粒plk0.1-puro/PTTG1。重组质粒以不同浓度转染,并于转染后不同时间收集细胞,以β-actin为内参,采用RT-PCR和Western blot技术分别检测PTTG1基因的mRNA、蛋白质的相对表达水平。结果:与空白对照组相比,6孔板中每孔转染质粒plk0.1-puro/PTTG 11000ng以上、转染超过12d时,PTTG1基因mRNA、蛋白质的相对表达水平下降70%以上。结论:重组质粒plk0.1-puro/PTTG1,在6孔细胞培养板中以1000ng/孔转染12d以上时,有效抑制了人PTTG1基因的表达,在关于PTTG1基因的研究中具有较好的应用价值。     

PTTG和血管生成与结直肠癌的相关性研究

目的研究垂体肿瘤转化基因(PTTG)和血管生成与结直肠癌生物学行为的关系,探讨PTTG的表达与结直肠癌新生血管的关系。方法应用免疫组织化学法,检测120例结直肠癌、30例正常结直肠黏膜中PTTG的表达情况,并测定这些组织中微血管密度(MVD),结合临床病理学指标进行统计学分析。结果 PTTG在结直肠癌患者中阳性表达率为84.17%,其阳性表达率显著高于对照组的6.67%(P0.01);其表达水平与患者Dukes分期及淋巴结转移有关(P0.05)。结直肠癌组织中MVD计数平均为34.75±5.15,显著高于对照组19.10±2.77(P0.01);并与结直肠癌的Dukes分期、有无淋巴结转移有关(P0.05)。PTTG表达阳性病例,其MVD计数普遍高于表达阴性病例;在表达阳性病例中,MVD计数又与其阳性表达强度有关(P0.05)。结论 PTTG表达及微血管密度与结直肠癌生物学特性关系密切;PTTG表达可能促进了结直肠癌肿瘤血管生成;联合检测PTTG和MVD可作为结直肠癌恶性程度及预后指标;抑制PTTG活性有可能为结直肠癌防治开辟新的前景。     

PTTG is a Secretory Protein in Human Pituitary Adenomas and in Mouse Pituitary Tumor Cell Lines

The pituitary tumor-transforming gene (PTTG) is a homolog of yeast Securin, which arrests the activation of Separin to induce sister chromatid separation in the transition from metaphase to anaphase. Pituitary tumor-transforming gene is also known to induce angiogenesis during pituitary tumorigenesis. It has not been clarified whether PTTG functions as a cytoplasmic or a nuclear protein. Our immunohistochemical study indicated that PTTG is localized in the cytoplasm of pituitary tumor cells. In the present study, confocal laser scanning microscopy (CLSM) analysis of human pituitary adenomas and immunoelectron microscopy of the mouse pituitary cell line, AtT-20, demonstrated the localization of PTTG in the Golgi apparatus and vesicles. Secreted PTTG was detected by immunoblotting from culture medium of mouse pituitary tumor cell lines. Our results suggested that PTTG is a secretory protein produced by pituitary tumor cells. In addition, PTTG may exert autocrine and/or paracrine functions as a newly proposed important pathway for the action of PTTG.     

基于TCGA、THPA、GEO数据库挖掘PTTG1在乳腺癌中的表达及预后意义

目的:通过对癌症基因组图谱(The Cancer Genome Atlas,TCGA)、人类蛋白组图谱(The Human Protein Atlas,THPA)和人类肿瘤相关的基因表达汇编(Gene Expression Omnibus,GEO)数据库的数据挖掘,探索垂体肿瘤转化基因1（pituitary tumor-transforming gene 1,PTTG1）在乳腺癌中的表达及预后意义。方法:从TCGA、THPA和GEO数据库提取PTTG1在乳腺癌及正常组织中mRNA和蛋白水平的变化和患者临床病理资料,GEO在线预后分析数据库Kaplan-Meier Plotter(http://kmplot.com)检索PTTG1乳腺癌预后的相关性。结果:在数据库中,PTTG1 mRNA和蛋白水平在乳腺癌队列中表达显著高于正常乳腺组织(P<0.01);PTTG1 mRNA在不同的分子亚型中表达存在显著差异(P<0.05);PTTG1 mRNA在ER和PR阳性乳腺癌患者组织中表达显著低于ER和PR阴性乳腺癌患者(P<0.05);PTTG1 mRNA在进展期乳腺癌患者组织中表达显著高于早期乳腺癌患者(P<0.05);PTTG1 mRNA随着乳腺癌患者组织学分级程度升高而降低(P<0.05)。PTTG1低表达乳腺癌患者的预后显著好于其高表达患者(P<0.001);PTTG1高表达的ER和PR阳性、HER-2阴性乳腺癌患者的预后均显著差于其低表达者(P<0.05)。PTTG1高表达乳腺癌样本富集到细胞因子受体相互作用、趋化因子信号通路、细胞周期、自然杀伤细胞介导细胞毒性、Toll样受体信号通路和T细胞受体信号通路等相关基因集。结论:PTTG1 mRNA和蛋白在乳腺癌组织中高表达,PTTG1 mRNA表达与乳腺癌预后相关且在不同分子分型的乳腺癌中具有不同的预后监测意义,可以作为乳腺癌预后判断的标志物。     

PTTG 基因对非霍奇金淋巴瘤细胞增殖能力的影响

目的：观察垂体瘤转化基因（pituitary tumor-transforming gene ,PTTG）对非霍奇金淋巴瘤细胞SUDHL-4增殖能力的影响。方法通过shRNA的方法在高表达PTTG的SUDHL-4细胞中敲降PTTG基因,通过MTT实验、细胞周期分析检测SUDHL-4生物学行为的变化。结果 PTTG被敲降后SUDHL-4细胞的生长速度明显下降,细胞G0／G1比例、S期比例下降;G2／M期比例升高,细胞阻滞于G2／M期,差异均有统计学意义（P＜0.05）。结论 PTTG基因可能在非霍奇金淋巴瘤的发生发展中起重要作用,可能成为判断非霍奇金淋巴瘤预后的分子标志物及临床治疗的靶点。     

RNAi-mediated knockdown of pituitary tumor-transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells

Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy.     

Expressions and their significance of PTTG and PC proteins in glioma

Objective： To investigate the expressions and their relationship of pituitary tumor transforming gene （PTTG） and proliferating cell nuclear antigen （PCNA） in glioma. Methods： The protein expressions of PTTG and PCNA were detected by immunostaining assay using streptavidin-peroxidase （SP） method in 80 cases of glioma. Results： The positive rates of PTTG in grades Ⅰ-Ⅳ gliomas were 56.3%, 68.2%, 80.8%, and 100.0% respectively, and the protein expression of PTTG increased with the increasing of the pathological grade （X^2= 9.602, P 〈 0.05）; The positive rates of PCNA protein were 37.5%, 54.5%, 69.2%, and 93.8% respectively, and the protein expression of PCNA increased with the increasing of the pathological grade （X2 = 12.147, P 〈 0.01）. The expression of PTTG had positive correlation with the expression of PCNA protein （~s = 0.557, P 〈 0.01）. Conclusion： The expressions of PTTG and PCNA proteins were related to malignant degree of glioma, and may cooperate with each other in the tumorigenesis and progression and can be considered as the indicators of the biological behaviors in glioma.     

PTEN、Survivin和PTTG与垂体腺瘤侵袭性的关系

目的探讨抑癌基因（PTEN）、凋亡抑制基因（survivin）、垂体腺瘤转化基因（PTTG）表达与垂体腺瘤侵袭性的关系。方法将49例垂体腺瘤手术标本分为侵袭组与非侵袭组，采用免疫组化SP法。检测PTEN、survivin、PTTG的表达。分析它们在侵袭组与非侵袭组中表达水平的差异性。结果侵袭组中的survivin、PTTG表达明显高于非侵袭组，PTEN表达则相反（P均〈0．05）。survivin与PTEN的表达强度呈负相关：PTTG与survivin的表达强度呈正相关，与PTEN的表达强度呈负相关。结论PTEN、survivin、PTTG表达与垂体腺瘤的侵袭性密切相关，对垂体腺瘤的发生发展起重要作用。     

PTTG表达与垂体腺瘤侵袭性关系

目的 探讨垂体瘤转化基因(Pituitary tumor transforming gene，PTTG)表达与腺瘤侵袭性的关系及其临床意义。方法 回顾近年我科经手术切除的44例垂体腺瘤标本及6例正常垂体组织，均经10％的福尔马林固定、石蜡包埋。联合运用Knosp影像学标准及任祖渊标准，参考术中情况，将肿瘤标本分为侵袭组与非侵袭组；运用免疫组化的方法检测这50例标本中PTTG蛋白表达情况；分析PTTG表达与垂体腺瘤侵袭性的内在关系。结果 免疫组化实验结果显示PTTG蛋白在侵袭性垂体腺瘤组中显著高表达，在非侵袭性腺瘤组中有一定表达，在正常垂体组织中未见表达。结论 原癌基因PTTG表达水平和垂体腺瘤侵袭性密切相关，可作为腺瘤侵袭性的标志物。     

PTTG基因在人乳腺癌中的原位表达及其意义

目的　研究PTTG基因在乳腺癌表达的临床病理意义。方法　应用原位杂交 (DNA RNA)技术检测 66例人乳腺癌、2 7例乳腺小叶增生及 5 4乳腺正常组织中PTTGmRNA。结果　PTTGmRNA在乳腺癌、乳腺小叶增生和乳腺正常组织阳性率分别为 71 2 % (4 7/66)、5 1 9% (14 /2 7)和 11 1% (6/5 4) ,乳腺癌和乳腺正常组织之间有显著差异 (P <0 0 5 ) ,乳腺小叶增生和乳腺正常组织之间也有显著差异 (P <0 0 5 ) ;PTTGmRNA的表达与淋巴结转移无统计学意义 (P >0 0 5 )。结论　PTTG基因的过度表达可能参与了人乳腺癌的发生发展过程