PTTG1在脑胶质瘤中的表达和microRNA靶向抑制PTTG1对脑胶质瘤细胞生物学特性影响的实验研究
|
摘要
脑胶质瘤是中枢神经系统最常见的恶性肿瘤,约占成人颅内肿瘤的35%～60%,其中恶性胶质瘤约占60%,预后差,病死率高。在胶质瘤的发生、发展中有多种基因的连锁改变,这些改变最终使肿瘤细胞获得无限增殖的生长优势,从而促进肿瘤的发生和发展。
垂体瘤转化基因PTTG,又称Securin,是一种致癌基因,是1997年Pei等人在老鼠脑垂体肿瘤GH4细胞系的研究中被发现的。PTTG1在许多肿瘤中高表达。其在肿瘤发生中作用机制涉及到细胞转化、非整数倍细胞分裂、细胞调亡和影响致瘤的微环境。目前国外仅有小宗脑胶质瘤标本的研究,尚无PTTG1基因表达与脑胶质瘤病理类型、增殖活性、生物学行为等方面的研究报道。为探讨PTTG1基因在胶质瘤发生发展中的作用,本课题通过对胶质瘤中PTTG1表达与胶质瘤恶性程度、肿瘤细胞增殖活性及其相互关系进行了研究,并通过体内、体外试验探讨PTTG1基因作为基因治疗胶质瘤优选靶的之一的可行性;初步探讨PTTG1基因影响脑胶质瘤生物学特性的可能机制。
1、人脑胶质瘤中PTTG1的表达和临床病理相关性分析
采用实时荧光定量RT-PCR方法和Western blot杂交检测PTTG1 mRNA和蛋白在正常脑组织、胶质瘤和恶性人脑胶质瘤细胞系的表达。结果表明:在正常脑组织中未检测到PTTG1 mRNA和蛋白的表达;在各级胶质瘤中均有PTTG1的表达,其表达强度与肿瘤恶性程度呈正相关关系;在Ⅲ-Ⅳ级(高恶度)胶质瘤中高表达,而在Ⅱ级(低恶度)胶质瘤中表达量较低,Ⅲ-Ⅳ级与Ⅱ级PTTG1 mRNA、蛋白的表达量相比较有显著差异(P＜0.001)。采用免疫组化ABC法检测了4例正常脑组织及52例人脑胶质瘤石蜡标本中PTTG1蛋白的表达情况。结果显示,正常脑胶质细胞中基本无PTTG1蛋白的表达,绝大部分星形细胞瘤标本中可见PTTG1蛋白的阳性表达,其表达情况为:Ⅱ级55%(11/20),Ⅲ级76.2%(11/15),Ⅳ级100%(17/17)。在Ⅱ、Ⅲ和Ⅳ级3个不同病理级别的星形细胞肿瘤中,PTTG1蛋白的阳性表达率有显著差别(P＜0.01)。大部分其它类型的胶质瘤中也可见PTTG1蛋白的阳性表达,其表达情况为:少突胶质细胞瘤75%(3/4),室管膜瘤100%(4/4)。脑胶质瘤的临床病理级别、分化程度与PTTG1的表达阳性率之间比较有统计学上的显著差异性(P＜0.01);而性别和年龄则无统计学上的差异(P＞0.05)。用Ki-67标记指数(Ki-67 LabelingIndex)评价胶质瘤的增殖活性,基质金属蛋白酶(matrix metalloproteinases,MMPs)对于肿瘤的侵袭具有极其重要的作用。本实验以免疫组化法检测了Ki-67、MMP2和MMP9的表达强度,Ki67 LI检验三种病理级别胶质瘤Ki67表达情况,其表达情况为:Ⅱ级(1.88),Ⅲ级(8.94),Ⅳ级(13.77),统计学上有差异(P＜0.001),说明Ki67 LI在不同级别的胶质瘤中具有显著性差异。39例胶质瘤中MMP2、MMP9蛋白阳性表达率分别是:Ⅱ级的纤维型星形细胞瘤阳性表达45%、40%,Ⅲ级的间变型星形细胞瘤80%、86.7%,Ⅳ级的多形性胶质母细胞瘤均为100%,统计学上有差异(P＜0.01)。胶质瘤中Ki-67LI、MMP2、MMP9的表达与PTTG1基因表达、肿瘤分级呈正相关,结果提示胶质瘤中PTTG1基因表达与肿瘤细胞增殖活性、侵袭性密切相关。
2、microRNA靶向性抑制PTTG1表达对人脑胶质瘤细胞体外增殖、凋亡以及侵袭性的影响
利用两个恶性胶质瘤体外细胞系U251和SHG-44为模型,将含靶向PTTG1的microRNA(miRNA)的质粒pcDNA~(TM)6.2-GW/EmGFPmiR通过Lipofectamine介导分别转染U251人脑胶质母细胞瘤细胞和SHG-44恶性星形细胞瘤细胞,通过荧光定量PCR、Western blot、激光共聚焦显微镜、MTT法、流式细胞术(FCM)以及Caspase-3检测定性和定量研究了miRNA-PTTG1抑制PTTG1的表达,以及了解对U251、SHG-44体外细胞增殖、凋亡以及侵袭性的影响。MTT法检测细胞增殖率,发现转染后的U251、SHG-44细胞克隆生长速度明显下降。采用FCM定量分析各组细胞的调亡水平,结果显示,与未转染的U251细胞(早期调亡细胞16.9%)和转染阴性质粒的U251细胞(早期调亡细胞14.9%)相比,转染MIR-2干扰质粒U251细胞中调亡细胞明显增多,48小时后调亡细胞达20.4%,统计学上有差异(P＜0.001);在SHG-44细胞实验中,与未转染的SHG-44细胞(早期调亡细胞15.2%)和转染阴性质粒的SHG-44细胞(早期调亡细胞14%)相比,转染MIR-2干扰质粒SHG-44细胞中调亡细胞明显增多,48小时后调亡细胞达22.2%,统计学上有差异(P＜0.001)。Caspase-3荧光活性检测也显示,转染miRNA-PTTG1的U251细胞和SHG-44细胞中Caspase-3的活性明显增加,与未转染的U251细胞(12.60%)和转染阴性质粒的U251细胞(28.04%)相比,转染MIR-2干扰质粒U251细胞中caspase-3的相对活性明显升高(56.37%),统计学上有差异(P＜0.001);与未转染的SHG-44细胞(27.13%)和转染阴性质粒的SHG-44细胞(29.25%)相比,转染MIR-2干扰质粒SHG-44细胞中caspase-3的相对活性明显升高(48.88%),统计学上有差异(P＜0.001)。Matrigel体外侵袭实验和细胞迁移运动实验中,转染miRNA-PTTG1的U251细胞与U251对照组和阴性载体干扰组比较侵袭能力分别下降了36.5%和30%,迁移能力下降了64.2%和64.7%。Western blot分析:miRNA干扰PTTG1在脑胶质瘤细胞中表达后,与细胞增殖相关的蛋白Ki-67、c-myc表达下调;细胞调亡相关的蛋白p53、Bax、p21WAF1/Cip1表达上调;与肿瘤细胞侵袭性相关的蛋白MMP2、MMP9表达下降(P＜0.05)。研究结果提示:Lipofectamine-pcDNA~(TM)6.2-GW/EmGFPmiR-PTTG1复合物可成功地将针对PTTG1的外源性miRNA导入至胶质瘤内,抑制肿瘤细胞的PTTG1基因表达,对PTTG1 mRNA及蛋白明显高表达的U251、SHG-44细胞的生长和增殖有明显的抑制作用,诱导胶质瘤细胞调亡和降低恶性胶质瘤细胞的侵袭性。结果提示PTTG1基因有可能成为基因治疗恶性胶质瘤的优选靶之一。
3、microRNA抑制PTTG1蛋白对人脑胶质瘤细胞裸鼠成瘤性的影响
在裸鼠成瘤试验中,我们将U251细胞(A组),U251+pcDNA~(TM)6.2-GW/EmGFPmiR-negative(转染阴性质粒U251细胞)细胞(B组)和U251+pcDNA~(TM)6.2-GW/EmGFPmiR-PTTG1(转染干扰质粒U251细胞)细胞(C组)分别接种于裸鼠背部皮下,每组5只裸鼠,建立人脑胶质瘤裸鼠皮下移植瘤模型。定期观察肿瘤生长情况,测定肿瘤体积,绘制肿瘤生长曲线,观察4周后处死动物,称瘤重。接种4周后,肉眼就可以看到,接种正常U251细胞的肿瘤明显大于其他两组。接种正常U251细胞的肿瘤明显大于其他两组。4周时测得的各组肿瘤体积分别为:Mock-U251组为(2.36±0.22)cm~3;Negative-U251组为(2.06±0.08)cm~3;Mir-2-U251组为(1.50±0.05)cm~3。处死后,切下3组肿瘤分别称重,Mock-U251组平均瘤重为(1.71±0.13)g,Negative-U251组为(1.43±0.19)g;Mir-2-U251组为(0.74±0.07)g,结果显示,与A组和B组裸鼠相比,C组裸鼠肿瘤形成时间延迟,肿瘤生长缓慢,肿瘤体积及瘤重均明显减小(P均＜0.05)。HE染色显示各组肿瘤组织符合多形性胶质母细胞瘤的形态,靶向抑制PTTG1的miRNA干扰治疗后出现了肿瘤中心坏死,坏死区细胞稀疏,向外细胞渐增多,其间含有很多的破碎细胞,再向外则为密集的瘤细胞,并有细胞内、外的水肿和出血,同时也有不同程度的核固缩细胞。Westernblot检测显示,与A组和B组相比,C组裸鼠肿瘤组织中活性的Capase-3蛋白明显上调(P＜0.001)。上述结果提示在裸鼠肿瘤细胞中miRNA可通过抑制PTTG1蛋白的表达来拮抗PTTG1蛋白抑制细胞凋亡的作用,并激活活性Capase-3蛋白表达,从而促进了肿瘤细胞的凋亡。
综上所述,本课题的结论是在胶质瘤组织中,PTTG1的蛋白表达水平随胶质瘤恶性程度的增加相应增高,PTTG1在恶性胶质瘤细胞系中高表达。PTTG1表达的升高与肿瘤分化和转移密切相关。人脑胶质瘤中PTTG1、MMP2、MMP9、Ki67的表达水平与胶质瘤的病理分级成正相关,提示在恶性胶质瘤中PTTG1与胶质瘤的恶性表型(增殖和侵袭)密切相关。microRNA干扰PTTG1表达后,western blot检测的细胞增殖密切相关的核抗原Ki67以及MAPK级联反应中的靶标c-myc的表达下调,MTT实验的结果提示抑制PTTG1表达后U251、SHG-44人脑胶质瘤细胞系的细胞增殖率均明显下降,都出现了生长减慢。利用基因沉默技术靶向PTTG1的microRNA表达载体有效地抑制了胶质瘤细胞系U251和SHG-44中PTTG1的表达,p53以及其下游调控基因p21WAF1/Cip1、Bax的表达都发生上调的变化,caspase-3的相对活性明显升高,从而导致P53功能上调引起Bax、P21表达上调从而激活caspase-3最终诱导胶质瘤细胞发生凋亡。PTTG1的RNA干扰可以明显逆转U251人脑胶质瘤细胞系的侵袭和迁移,MMP2、MMP9 Western杂交结果显示表达显著减少,提示PTTG1→bFGF→MMP→肿瘤细胞基底膜屏障突破这一调节通路可能是恶性脑胶质瘤侵袭性产生的重要机制之一。转染外源性microRNA抑制PTTG1基因的U251细胞在无免疫裸鼠皮下成瘤后,细胞生长减慢,胶质瘤组织细胞出现增殖抑制、凋亡增加。体内外实验证明:证实了PTTG1蛋白可能是人脑胶质瘤形成和发展过程中的一个关键蛋白,其作用机制可能是PTTG1蛋白通过促进细胞增殖、抑制肿瘤细胞凋亡促进脑胶质瘤细胞的发生和发展,而且PTTG1和肿瘤细胞侵袭性密切相关。以PTTG1蛋白为靶点来设计新的脑胶质瘤基因治疗方法是很有前景的。
Glioma is the most common type of primary intracranial tumors.It has been reported that malignant glioma accounted for about 60%of gliomas.Although the currently available multi-modalities of therapies,such as microsurgical operation,radiotherapy and chemotherapy,have been improved,the median survival time of patients with malignant glioma is only 52 weeks.So glioma is still a refractory disease in the neurosurgical field.In order to improye the present situation,the mechanism of the development and progression of gliomas as well as the new strategies for the treatment of malignant gliomas should be studied in the future.With the significant advances of molecular biology,it has been demonstrated that the development of malignant tumors including gliomas is due to the activation of proto-oncogenes and inactivation of tumor suppressor genes.PTTG was isolated from rat pituitary tumor cells in 1997 and identified as a pituitary-derived transforming gene.Recent studies have shown that PTTG1 gene is overexpressed in a variety of tumors.Forced PTTG1 expression induces cell transformation in vitro and tumor formation in nude mice.In some tumors,high PTTG1 levels correlate with invasiveness, and PTTG1 has been identified as a key signature gene associated with tumor metastasis. Increasing evidence supports a multifunctional role of PTTG1 in cell physiology and tumorigenesis.Physiological PTTG1 properties include securin activity,DNA damage/repair regulation and involvement in organ development and metabolism. Tumorigenic mechanisms for PTTG1 action involve cell transformation and aneuploidy, apoptosis,and tumorigenic microenvironment feedback.So PTTG1 gene may play an important role in the malignant progression in gliomas.Recent knowledge gained from PTTG1-null mouse models and transgenic animals and their potential application to subcellular therapeutic targeting PTTG1 are discussed.Up to date,although there have been some reports on the expression of PTTG1 genes in gliomas,but these results were obtained from a small number of brain tumors or glioma cell lines,the relationship among PTTG1 gene expression,histopathological type,proliferation activity,biological behavior of gliomas has not been reported.In the present study,the expression of PTTG1 gene in gliomas and the relationship among PTTG1 gene expression,the tumor grade and tumor proliferation were investigated in order to understand the role of PTTG1 gene in the pathogenesis of gliomas.The feasibility of using PTTG1 gene as a target for gene therapy of gliomas was also studied by in vitor and in vivo experiments.
PartⅠ:Study on the gene expression of PTTG1 and its relationship to clinic histopathologic of human gliomas
The gene expression of PTTG1 in 52 human gliomas,2 malignant human glioma cell lines and 4 normal brain tissues were studied by real-time RT-PCR and western blot analysis.It was found that 4 normal brain tissues almost no expressed PTTG1 and its mRNA,and 2 malignant human glioma cell lines had PTTG1 and its mRNA overexpression.The difference of positive rate and expression level of PTTG1 and its mRNA between the low-grade and high-grade tumors is statisticly significant(P＜0.05). The results of Western blot analysis using antibody performed on 52 samples of human gliomas were coincidence with that of immunohistochemical staining(P＜0.01).The expression level of PTTG1 gene also provide us a useful parameter in evaluating the degree of malignancy in molecular level and selecting the target of gene therapy. Relationship between proliferation activity and PTTG1 gene expression in human gliomas was found that the proliferation activity of gliomas evaluated by Ki-67 LI(Ki-67 Labeling Index) is positively correlated with PTTG1 gene expression,and positively with the tumor grade.These results suggested that PTTG1 gene expression was significantly associated with the proliferation activity in human gliomas.And PTTG1 gene expression in human gliomas was found that is positively correlated with MMP2 and MMP9 gene expression.
PartⅡ:Study of the effect of transfected miRNA-PTTG1 on proliferation,apoptosis and invasion of glioma cells in vitro
The pcDNA~(TM) 6.2-GW/EmGFPmiR-PTTG1 plasmid which contain the specific miRNA trageting PTTG1 and the negative plasmid pcDNA~(TM) 6.2-GW/EmGFP miR-negative were transfected respectively into human brain glioblastoma U251 and SHG-44 cells by lipofectin medium.The impact on proliferation and apoptosis of U251, SHG-44 cells in vitro after inhibition of PTTG1 with miRNA was investigated by methods of real-time RT-PCR,western-blot,Confocal Scanning Laser Microscope(CSLM),flow cytometry(FCM) and caspase-3 activity assay.The U251 glioblastoma cell transfectants and the SHG-44 astrocytoma cell ones resulted in dramatic down-regulation of PTTG1 mRNA and protein as demonstrated by real-time RT-PCR,western-blot analysis.Four clones with low expression of PTTG1 constructal so showed decreased proliferation in vitro as detected by MTT method.The quantitative analysis of apoptotic cells by FCM showed that the apoptotic cells increased in U251 cells(tranfected with miRNA-PTTG1) with the apoptotic rate of 20.4%compared with U251(16.9%) and U251 cells(tranfected with pcDNA~(TM) 6.2-GW/EmGFPmiR-negative)(14.9%).And the quantitative,analysis of apoptotic cells by FCM showed that the apoptotic cells increased in SHG-44 cells(tranfected with miRNA-PTTG1) with the apoptotic rate of 22.2%compared with SHG-44(15.2%) and SHG-44 cells(tranfected with pcDNA~(TM) 6.2-GW/EmGFPmiR-negative)(14%).Caspase-3 activity assay indicated that the activated caspase-3 was significantly increased(P＜0.01) in U251 and SHG-44 cells (tranfected with miRNA-PTTG1).Transient transfection of U251 and SHG-44 cells with miRNA-PTTG1 showed a significant increase in expression of p53,Bax and p21WAF1/Cip1,decrease in expression of and ki67,c-myc,p53,MMP2,MMP9 measured by western blot analysis.And the results by FCM also showed that RNA knockdown can sensitize glioma cells to p53-dependent apoptosis.The above results suggested that by miRNA can interfere with PTTG1 expression,and induce apoptotic cell death in glioma cells,and tumor suppressor protein p53 is required for miRNA-PTTG induced apoptotic cell death,and inhibition of PTTG1 could be a useful therapeutic strategy for the treatment of glioma.The effect on invasiveness of U251 cells was investigated by methods of Matrigel and wound-healing.And the results showed that RNA knockdown of PTTG1 can inhibit the invasiveness of glioma.cells.PTTG1 gene may be selected as a target for gene therapy of gilomas,and it should be studied further in vivo experiment.
PartⅢ:The specific therapeutic implication of miRNA-PTTG1 in vivo for xenograft gliomas
In order to further investigate the role of PTTG1 gene in the growth and proliferation of glioma cells,and find out the feasibility of using PTTG1 gene as a target for gene therapy of gliomas as well as to observe the therapeutic effect of PTTG1 gene for gliomas in vivo and it's prospect in clinical application.15 nude mices weighing 16～20g were divided into three groups:U251 cells(A group),U251 plus negative vector-treated group(transfected with transfected with pcDNA~(TM) 6.2-GW/Em GFPmiR-negative)(B group),U251 plus miRNA-PTTG1-treated group(transfected with pcDNA~(TM) 6.2-GW/EmGFPmiR-PTTG1)(C group) were inoculated respectively in back subcutaneous tissue of nude mice to establish xenograft models of human brain glioma. The tumor growth status was observed and tumor volume was measured termly,and the tumor growth curve was drawn.The animals were killed and the tumor weight was investigated 4 weeks after inoculation.The results showed that the tumorigenesis time delayed,tumor grew slow,both tumor volume and tumor weight decreased significantly (P＜0.05) in C group as compared with those in A and B groups.HE staining for each group of tumor specimen indicated thatthe histological features of these nude mice tumor is in accordance with those of glioblastoma multiformes.The results of western blot showed that the activated caspase-3 was significantly increased in C group in comparison with those in A and B groups.The above results suggested that by disrupting PTTG1 expression,miRNA mediated RNA knockdown can antagonize the anti-apoptotic effect of PTTG1,activate caspase-3,and then induce apoptotic death of U251 cells in nude mice human brain glioma.
In summary,this research suggested that PTTG1 may be a key protein in initiation and progress of human brain glioma,its mechanism may involve the proliferative and anti-apoptotic effect of PTTG1 proteins in the tumorigenesis and development of glioma. Blocking of function of PTTG1 or down regulation of its expression in tumors may result in suppression of tumor growth and metastasis.PTTG1 may be considered as a potential and promising target for gene therapy of glioma.
垂体瘤转化基因PTTG,又称Securin,是一种致癌基因,是1997年Pei等人在老鼠脑垂体肿瘤GH4细胞系的研究中被发现的。PTTG1在许多肿瘤中高表达。其在肿瘤发生中作用机制涉及到细胞转化、非整数倍细胞分裂、细胞调亡和影响致瘤的微环境。目前国外仅有小宗脑胶质瘤标本的研究,尚无PTTG1基因表达与脑胶质瘤病理类型、增殖活性、生物学行为等方面的研究报道。为探讨PTTG1基因在胶质瘤发生发展中的作用,本课题通过对胶质瘤中PTTG1表达与胶质瘤恶性程度、肿瘤细胞增殖活性及其相互关系进行了研究,并通过体内、体外试验探讨PTTG1基因作为基因治疗胶质瘤优选靶的之一的可行性;初步探讨PTTG1基因影响脑胶质瘤生物学特性的可能机制。
1、人脑胶质瘤中PTTG1的表达和临床病理相关性分析
采用实时荧光定量RT-PCR方法和Western blot杂交检测PTTG1 mRNA和蛋白在正常脑组织、胶质瘤和恶性人脑胶质瘤细胞系的表达。结果表明:在正常脑组织中未检测到PTTG1 mRNA和蛋白的表达;在各级胶质瘤中均有PTTG1的表达,其表达强度与肿瘤恶性程度呈正相关关系;在Ⅲ-Ⅳ级(高恶度)胶质瘤中高表达,而在Ⅱ级(低恶度)胶质瘤中表达量较低,Ⅲ-Ⅳ级与Ⅱ级PTTG1 mRNA、蛋白的表达量相比较有显著差异(P＜0.001)。采用免疫组化ABC法检测了4例正常脑组织及52例人脑胶质瘤石蜡标本中PTTG1蛋白的表达情况。结果显示,正常脑胶质细胞中基本无PTTG1蛋白的表达,绝大部分星形细胞瘤标本中可见PTTG1蛋白的阳性表达,其表达情况为:Ⅱ级55%(11/20),Ⅲ级76.2%(11/15),Ⅳ级100%(17/17)。在Ⅱ、Ⅲ和Ⅳ级3个不同病理级别的星形细胞肿瘤中,PTTG1蛋白的阳性表达率有显著差别(P＜0.01)。大部分其它类型的胶质瘤中也可见PTTG1蛋白的阳性表达,其表达情况为:少突胶质细胞瘤75%(3/4),室管膜瘤100%(4/4)。脑胶质瘤的临床病理级别、分化程度与PTTG1的表达阳性率之间比较有统计学上的显著差异性(P＜0.01);而性别和年龄则无统计学上的差异(P＞0.05)。用Ki-67标记指数(Ki-67 LabelingIndex)评价胶质瘤的增殖活性,基质金属蛋白酶(matrix metalloproteinases,MMPs)对于肿瘤的侵袭具有极其重要的作用。本实验以免疫组化法检测了Ki-67、MMP2和MMP9的表达强度,Ki67 LI检验三种病理级别胶质瘤Ki67表达情况,其表达情况为:Ⅱ级(1.88),Ⅲ级(8.94),Ⅳ级(13.77),统计学上有差异(P＜0.001),说明Ki67 LI在不同级别的胶质瘤中具有显著性差异。39例胶质瘤中MMP2、MMP9蛋白阳性表达率分别是:Ⅱ级的纤维型星形细胞瘤阳性表达45%、40%,Ⅲ级的间变型星形细胞瘤80%、86.7%,Ⅳ级的多形性胶质母细胞瘤均为100%,统计学上有差异(P＜0.01)。胶质瘤中Ki-67LI、MMP2、MMP9的表达与PTTG1基因表达、肿瘤分级呈正相关,结果提示胶质瘤中PTTG1基因表达与肿瘤细胞增殖活性、侵袭性密切相关。
2、microRNA靶向性抑制PTTG1表达对人脑胶质瘤细胞体外增殖、凋亡以及侵袭性的影响
利用两个恶性胶质瘤体外细胞系U251和SHG-44为模型,将含靶向PTTG1的microRNA(miRNA)的质粒pcDNA~(TM)6.2-GW/EmGFPmiR通过Lipofectamine介导分别转染U251人脑胶质母细胞瘤细胞和SHG-44恶性星形细胞瘤细胞,通过荧光定量PCR、Western blot、激光共聚焦显微镜、MTT法、流式细胞术(FCM)以及Caspase-3检测定性和定量研究了miRNA-PTTG1抑制PTTG1的表达,以及了解对U251、SHG-44体外细胞增殖、凋亡以及侵袭性的影响。MTT法检测细胞增殖率,发现转染后的U251、SHG-44细胞克隆生长速度明显下降。采用FCM定量分析各组细胞的调亡水平,结果显示,与未转染的U251细胞(早期调亡细胞16.9%)和转染阴性质粒的U251细胞(早期调亡细胞14.9%)相比,转染MIR-2干扰质粒U251细胞中调亡细胞明显增多,48小时后调亡细胞达20.4%,统计学上有差异(P＜0.001);在SHG-44细胞实验中,与未转染的SHG-44细胞(早期调亡细胞15.2%)和转染阴性质粒的SHG-44细胞(早期调亡细胞14%)相比,转染MIR-2干扰质粒SHG-44细胞中调亡细胞明显增多,48小时后调亡细胞达22.2%,统计学上有差异(P＜0.001)。Caspase-3荧光活性检测也显示,转染miRNA-PTTG1的U251细胞和SHG-44细胞中Caspase-3的活性明显增加,与未转染的U251细胞(12.60%)和转染阴性质粒的U251细胞(28.04%)相比,转染MIR-2干扰质粒U251细胞中caspase-3的相对活性明显升高(56.37%),统计学上有差异(P＜0.001);与未转染的SHG-44细胞(27.13%)和转染阴性质粒的SHG-44细胞(29.25%)相比,转染MIR-2干扰质粒SHG-44细胞中caspase-3的相对活性明显升高(48.88%),统计学上有差异(P＜0.001)。Matrigel体外侵袭实验和细胞迁移运动实验中,转染miRNA-PTTG1的U251细胞与U251对照组和阴性载体干扰组比较侵袭能力分别下降了36.5%和30%,迁移能力下降了64.2%和64.7%。Western blot分析:miRNA干扰PTTG1在脑胶质瘤细胞中表达后,与细胞增殖相关的蛋白Ki-67、c-myc表达下调;细胞调亡相关的蛋白p53、Bax、p21WAF1/Cip1表达上调;与肿瘤细胞侵袭性相关的蛋白MMP2、MMP9表达下降(P＜0.05)。研究结果提示:Lipofectamine-pcDNA~(TM)6.2-GW/EmGFPmiR-PTTG1复合物可成功地将针对PTTG1的外源性miRNA导入至胶质瘤内,抑制肿瘤细胞的PTTG1基因表达,对PTTG1 mRNA及蛋白明显高表达的U251、SHG-44细胞的生长和增殖有明显的抑制作用,诱导胶质瘤细胞调亡和降低恶性胶质瘤细胞的侵袭性。结果提示PTTG1基因有可能成为基因治疗恶性胶质瘤的优选靶之一。
3、microRNA抑制PTTG1蛋白对人脑胶质瘤细胞裸鼠成瘤性的影响
在裸鼠成瘤试验中,我们将U251细胞(A组),U251+pcDNA~(TM)6.2-GW/EmGFPmiR-negative(转染阴性质粒U251细胞)细胞(B组)和U251+pcDNA~(TM)6.2-GW/EmGFPmiR-PTTG1(转染干扰质粒U251细胞)细胞(C组)分别接种于裸鼠背部皮下,每组5只裸鼠,建立人脑胶质瘤裸鼠皮下移植瘤模型。定期观察肿瘤生长情况,测定肿瘤体积,绘制肿瘤生长曲线,观察4周后处死动物,称瘤重。接种4周后,肉眼就可以看到,接种正常U251细胞的肿瘤明显大于其他两组。接种正常U251细胞的肿瘤明显大于其他两组。4周时测得的各组肿瘤体积分别为:Mock-U251组为(2.36±0.22)cm~3;Negative-U251组为(2.06±0.08)cm~3;Mir-2-U251组为(1.50±0.05)cm~3。处死后,切下3组肿瘤分别称重,Mock-U251组平均瘤重为(1.71±0.13)g,Negative-U251组为(1.43±0.19)g;Mir-2-U251组为(0.74±0.07)g,结果显示,与A组和B组裸鼠相比,C组裸鼠肿瘤形成时间延迟,肿瘤生长缓慢,肿瘤体积及瘤重均明显减小(P均＜0.05)。HE染色显示各组肿瘤组织符合多形性胶质母细胞瘤的形态,靶向抑制PTTG1的miRNA干扰治疗后出现了肿瘤中心坏死,坏死区细胞稀疏,向外细胞渐增多,其间含有很多的破碎细胞,再向外则为密集的瘤细胞,并有细胞内、外的水肿和出血,同时也有不同程度的核固缩细胞。Westernblot检测显示,与A组和B组相比,C组裸鼠肿瘤组织中活性的Capase-3蛋白明显上调(P＜0.001)。上述结果提示在裸鼠肿瘤细胞中miRNA可通过抑制PTTG1蛋白的表达来拮抗PTTG1蛋白抑制细胞凋亡的作用,并激活活性Capase-3蛋白表达,从而促进了肿瘤细胞的凋亡。
综上所述,本课题的结论是在胶质瘤组织中,PTTG1的蛋白表达水平随胶质瘤恶性程度的增加相应增高,PTTG1在恶性胶质瘤细胞系中高表达。PTTG1表达的升高与肿瘤分化和转移密切相关。人脑胶质瘤中PTTG1、MMP2、MMP9、Ki67的表达水平与胶质瘤的病理分级成正相关,提示在恶性胶质瘤中PTTG1与胶质瘤的恶性表型(增殖和侵袭)密切相关。microRNA干扰PTTG1表达后,western blot检测的细胞增殖密切相关的核抗原Ki67以及MAPK级联反应中的靶标c-myc的表达下调,MTT实验的结果提示抑制PTTG1表达后U251、SHG-44人脑胶质瘤细胞系的细胞增殖率均明显下降,都出现了生长减慢。利用基因沉默技术靶向PTTG1的microRNA表达载体有效地抑制了胶质瘤细胞系U251和SHG-44中PTTG1的表达,p53以及其下游调控基因p21WAF1/Cip1、Bax的表达都发生上调的变化,caspase-3的相对活性明显升高,从而导致P53功能上调引起Bax、P21表达上调从而激活caspase-3最终诱导胶质瘤细胞发生凋亡。PTTG1的RNA干扰可以明显逆转U251人脑胶质瘤细胞系的侵袭和迁移,MMP2、MMP9 Western杂交结果显示表达显著减少,提示PTTG1→bFGF→MMP→肿瘤细胞基底膜屏障突破这一调节通路可能是恶性脑胶质瘤侵袭性产生的重要机制之一。转染外源性microRNA抑制PTTG1基因的U251细胞在无免疫裸鼠皮下成瘤后,细胞生长减慢,胶质瘤组织细胞出现增殖抑制、凋亡增加。体内外实验证明:证实了PTTG1蛋白可能是人脑胶质瘤形成和发展过程中的一个关键蛋白,其作用机制可能是PTTG1蛋白通过促进细胞增殖、抑制肿瘤细胞凋亡促进脑胶质瘤细胞的发生和发展,而且PTTG1和肿瘤细胞侵袭性密切相关。以PTTG1蛋白为靶点来设计新的脑胶质瘤基因治疗方法是很有前景的。
Glioma is the most common type of primary intracranial tumors.It has been reported that malignant glioma accounted for about 60%of gliomas.Although the currently available multi-modalities of therapies,such as microsurgical operation,radiotherapy and chemotherapy,have been improved,the median survival time of patients with malignant glioma is only 52 weeks.So glioma is still a refractory disease in the neurosurgical field.In order to improye the present situation,the mechanism of the development and progression of gliomas as well as the new strategies for the treatment of malignant gliomas should be studied in the future.With the significant advances of molecular biology,it has been demonstrated that the development of malignant tumors including gliomas is due to the activation of proto-oncogenes and inactivation of tumor suppressor genes.PTTG was isolated from rat pituitary tumor cells in 1997 and identified as a pituitary-derived transforming gene.Recent studies have shown that PTTG1 gene is overexpressed in a variety of tumors.Forced PTTG1 expression induces cell transformation in vitro and tumor formation in nude mice.In some tumors,high PTTG1 levels correlate with invasiveness, and PTTG1 has been identified as a key signature gene associated with tumor metastasis. Increasing evidence supports a multifunctional role of PTTG1 in cell physiology and tumorigenesis.Physiological PTTG1 properties include securin activity,DNA damage/repair regulation and involvement in organ development and metabolism. Tumorigenic mechanisms for PTTG1 action involve cell transformation and aneuploidy, apoptosis,and tumorigenic microenvironment feedback.So PTTG1 gene may play an important role in the malignant progression in gliomas.Recent knowledge gained from PTTG1-null mouse models and transgenic animals and their potential application to subcellular therapeutic targeting PTTG1 are discussed.Up to date,although there have been some reports on the expression of PTTG1 genes in gliomas,but these results were obtained from a small number of brain tumors or glioma cell lines,the relationship among PTTG1 gene expression,histopathological type,proliferation activity,biological behavior of gliomas has not been reported.In the present study,the expression of PTTG1 gene in gliomas and the relationship among PTTG1 gene expression,the tumor grade and tumor proliferation were investigated in order to understand the role of PTTG1 gene in the pathogenesis of gliomas.The feasibility of using PTTG1 gene as a target for gene therapy of gliomas was also studied by in vitor and in vivo experiments.
PartⅠ:Study on the gene expression of PTTG1 and its relationship to clinic histopathologic of human gliomas
The gene expression of PTTG1 in 52 human gliomas,2 malignant human glioma cell lines and 4 normal brain tissues were studied by real-time RT-PCR and western blot analysis.It was found that 4 normal brain tissues almost no expressed PTTG1 and its mRNA,and 2 malignant human glioma cell lines had PTTG1 and its mRNA overexpression.The difference of positive rate and expression level of PTTG1 and its mRNA between the low-grade and high-grade tumors is statisticly significant(P＜0.05). The results of Western blot analysis using antibody performed on 52 samples of human gliomas were coincidence with that of immunohistochemical staining(P＜0.01).The expression level of PTTG1 gene also provide us a useful parameter in evaluating the degree of malignancy in molecular level and selecting the target of gene therapy. Relationship between proliferation activity and PTTG1 gene expression in human gliomas was found that the proliferation activity of gliomas evaluated by Ki-67 LI(Ki-67 Labeling Index) is positively correlated with PTTG1 gene expression,and positively with the tumor grade.These results suggested that PTTG1 gene expression was significantly associated with the proliferation activity in human gliomas.And PTTG1 gene expression in human gliomas was found that is positively correlated with MMP2 and MMP9 gene expression.
PartⅡ:Study of the effect of transfected miRNA-PTTG1 on proliferation,apoptosis and invasion of glioma cells in vitro
The pcDNA~(TM) 6.2-GW/EmGFPmiR-PTTG1 plasmid which contain the specific miRNA trageting PTTG1 and the negative plasmid pcDNA~(TM) 6.2-GW/EmGFP miR-negative were transfected respectively into human brain glioblastoma U251 and SHG-44 cells by lipofectin medium.The impact on proliferation and apoptosis of U251, SHG-44 cells in vitro after inhibition of PTTG1 with miRNA was investigated by methods of real-time RT-PCR,western-blot,Confocal Scanning Laser Microscope(CSLM),flow cytometry(FCM) and caspase-3 activity assay.The U251 glioblastoma cell transfectants and the SHG-44 astrocytoma cell ones resulted in dramatic down-regulation of PTTG1 mRNA and protein as demonstrated by real-time RT-PCR,western-blot analysis.Four clones with low expression of PTTG1 constructal so showed decreased proliferation in vitro as detected by MTT method.The quantitative analysis of apoptotic cells by FCM showed that the apoptotic cells increased in U251 cells(tranfected with miRNA-PTTG1) with the apoptotic rate of 20.4%compared with U251(16.9%) and U251 cells(tranfected with pcDNA~(TM) 6.2-GW/EmGFPmiR-negative)(14.9%).And the quantitative,analysis of apoptotic cells by FCM showed that the apoptotic cells increased in SHG-44 cells(tranfected with miRNA-PTTG1) with the apoptotic rate of 22.2%compared with SHG-44(15.2%) and SHG-44 cells(tranfected with pcDNA~(TM) 6.2-GW/EmGFPmiR-negative)(14%).Caspase-3 activity assay indicated that the activated caspase-3 was significantly increased(P＜0.01) in U251 and SHG-44 cells (tranfected with miRNA-PTTG1).Transient transfection of U251 and SHG-44 cells with miRNA-PTTG1 showed a significant increase in expression of p53,Bax and p21WAF1/Cip1,decrease in expression of and ki67,c-myc,p53,MMP2,MMP9 measured by western blot analysis.And the results by FCM also showed that RNA knockdown can sensitize glioma cells to p53-dependent apoptosis.The above results suggested that by miRNA can interfere with PTTG1 expression,and induce apoptotic cell death in glioma cells,and tumor suppressor protein p53 is required for miRNA-PTTG induced apoptotic cell death,and inhibition of PTTG1 could be a useful therapeutic strategy for the treatment of glioma.The effect on invasiveness of U251 cells was investigated by methods of Matrigel and wound-healing.And the results showed that RNA knockdown of PTTG1 can inhibit the invasiveness of glioma.cells.PTTG1 gene may be selected as a target for gene therapy of gilomas,and it should be studied further in vivo experiment.
PartⅢ:The specific therapeutic implication of miRNA-PTTG1 in vivo for xenograft gliomas
In order to further investigate the role of PTTG1 gene in the growth and proliferation of glioma cells,and find out the feasibility of using PTTG1 gene as a target for gene therapy of gliomas as well as to observe the therapeutic effect of PTTG1 gene for gliomas in vivo and it's prospect in clinical application.15 nude mices weighing 16～20g were divided into three groups:U251 cells(A group),U251 plus negative vector-treated group(transfected with transfected with pcDNA~(TM) 6.2-GW/Em GFPmiR-negative)(B group),U251 plus miRNA-PTTG1-treated group(transfected with pcDNA~(TM) 6.2-GW/EmGFPmiR-PTTG1)(C group) were inoculated respectively in back subcutaneous tissue of nude mice to establish xenograft models of human brain glioma. The tumor growth status was observed and tumor volume was measured termly,and the tumor growth curve was drawn.The animals were killed and the tumor weight was investigated 4 weeks after inoculation.The results showed that the tumorigenesis time delayed,tumor grew slow,both tumor volume and tumor weight decreased significantly (P＜0.05) in C group as compared with those in A and B groups.HE staining for each group of tumor specimen indicated thatthe histological features of these nude mice tumor is in accordance with those of glioblastoma multiformes.The results of western blot showed that the activated caspase-3 was significantly increased in C group in comparison with those in A and B groups.The above results suggested that by disrupting PTTG1 expression,miRNA mediated RNA knockdown can antagonize the anti-apoptotic effect of PTTG1,activate caspase-3,and then induce apoptotic death of U251 cells in nude mice human brain glioma.
In summary,this research suggested that PTTG1 may be a key protein in initiation and progress of human brain glioma,its mechanism may involve the proliferative and anti-apoptotic effect of PTTG1 proteins in the tumorigenesis and development of glioma. Blocking of function of PTTG1 or down regulation of its expression in tumors may result in suppression of tumor growth and metastasis.PTTG1 may be considered as a potential and promising target for gene therapy of glioma.
引文
1. Pei L, Melmed S. Isolation and characterization of a pituitary tumor-transforming gene (PTTG). Mol Endocrinol 1997;11(4):433-41.
2. Zhang X, Horwitz GA, Prezant TR, Valentini A, Nakashima M, Bronstein MD, et al. Structure, expression, and function of human pituitary tumor-transforming gene (PTTG). Mol Endocrinol 1999;13(1):156-66.
3. Chen L, Puri R, Lefkowitz EJ, Kakar SS. Identification of the human pituitary tumor transforming gene (hPTTG) family: molecular structure, expression, and chromosomal localization. Gene 2000;248(1-2):41-50.
4. Saez C, Japon MA, Ramos-Morales F, Romero F, Segura DI, Tortolero M, et al. hpttg is over-expressed in pituitary adenomas and other primary epithelial neoplasias. Oncogene 1999;18(39):5473-6.
5. Filippella M, Galland F, Kujas M, Young J, Faggiano A, Lombardi G, et al. Pituitary tumour transforming gene (PTTG) expression correlates with the proliferative activity and recurrence status of pituitary adenomas: a clinical and immunohistochemical study. Clin Endocrinol (Oxf) 2006;65(4):536-43.
6. Heaney AP, Nelson V, Fernando M, Horwitz G. Transforming events in thyroid tumorigenesis and their association with follicular lesions. J Clin Endocrinol Metab 2001;86(10):5025-32.
7. Kakar SS, Jennes L. Molecular cloning and characterization of the tumor transforming gene (TUTR1): a novel gene in human tumorigenesis. Cytogenet Cell Genet 1999;84(3-4):211-6.
8. Thompson AD 3rd, Kakar SS. Insulin and IGF-I regulate the expression of the pituitary tumor transforming gene (PTTG) in breast tumor cells. FEBS Lett 2005;579(14):3195-200.
9. Tsai SJ, Lin SJ, Cheng YM, Chen HM, Wing LY. Expression and functional analysis of pituitary tumor transforming gene-1 [corrected] in uterine leiomyomas. J Clin Endocrinol Metab 2005;90(6):3715-23.
10. Kakar SS, Malik MT. Suppression of lung cancer with siRNA targeting PTTG. Int J Oncol 2006;29(2):387-95.
11. Rehfeld N, Geddert H, Atamna A, Rohrbeck A, Garcia G, Kliszewski S, et al. The influence of the pituitary tumor transforming gene-1 (PTTG-1) on survival of patients with small cell lung cancer and non-small cell lung cancer. J Carcinog 2006;5:4.
12. Mu YM, Oba K, Yanase T, Ito T, Ashida K, Goto K, et al. Human pituitary tumor transforming gene (hPTTG) inhibits human lung cancer A549 cell growth through activation of p21(WAF1/CIP1 ). Endocr J 2003;50(6):771-81.
13. Honda S. Hayashi M, Kobayashi Y, Ishikawa Y, Nakagawa K, Tsuchiya E. A role for the pituitary tumor-transforming gene in the genesis and progression of non-small cell lung carcinomas. Anticancer Res 2003;23(5A):3775-82.
14. Akino K, Akita S, Mizuguchi T, Takumi I, Yu R, Wang XY, et al. A novel molecular marker of pituitary tumor transforming gene involves in a rat liver regeneration. J Surg Res 2005;129(1): 142-6.
15. Cho-Rok J, Yoo J, Jang YJ, Kim S, Chu IS, Yeom YI, et al. Adenovirus-mediated transfer of siRNA against PTTGl inhibits liver cancer cell growth in vitro and in vivo. Hepatology 2006;43(5): 1042-52.
16. Zhou C, Liu S, Zhou X, Xue L, Quan L, Lu N, et al. Overexpression of human pituitary tumor transforming gene (hPTTG), is regulated by beta-catenin /TCF pathway in human esophageal squamous cell carcinoma. Int J Cancer 2005; 113(6):891-8.
17. Heaney AP, Singson R, McCabe CJ, Nelson V, Nakashima M, Melmed S. Expression of pituitary-tumour transforming gene in colorectal tumours. Lancet 2000;355(9205):716-9.
18. Chamaon K, Kirches E, Kanakis D, Braeuninger S, Dietzmann K, Mawrin C. Regulation of the pituitary tumor transforming gene by insulin-like-growth factor-I and insulin differs between malignant and non-neoplastic astrocytes. Biochem Biophys Res Commun 2005;331(1):86-92.
19. Tfelt-Hansen J, Yano S, Bandyopadhyay S, Carroll R, Brown EM, Chattopadhyay N. Expression of pituitary tumor transforming gene (PTTG) and its binding protein in human astrocytes and astrocytoma cells: function and regulation of PTTG in U87 astrocytoma cells. Endocrinology 2004;145(9):4222-31.
20. Wang Z, Melmed S. Pituitary tumor transforming gene (PTTG) transforming and transactivation activity. J Biol Chem 2000;275(11):7459-61.
21. Ramos-Morales F, Dominguez A, Romero F, Luna R, Multon MC, Pintor-Toro JA, et al. Cell cycle regulated expression and phosphorylation of hpttg proto-oncogene product. Oncogene 2000;19(3):403-9.
22. Yu R, Ren SG, Horwitz GA, Wang Z, Melmed S. Pituitary tumor transforming gene (PTTG) regulates placental JEG-3 cell division and survival: evidence from live cell imaging. Mol Endocrinol 2000; 14(8): 1137-46.
23. Zou H, McGarry TJ, Bernal T, Kirschner MW. Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis. Science 1999;285(5426):418-22.
24. Romero F, Multon MC, Ramos-Morales F, Dominguez A, Bernal JA, Pintor-Toro JA, et al. Human securin, hPTTG, is associated with Ku heterodimer, the regulatory subunit of the DNA-dependent protein kinase. Nucleic Acids Res 2001 ;29(6): 1300-7.
25. Dominguez A, Ramos-Morales F, Romero F, Rios RM, Dreyfus F, Tortolero M, et al. hpttg, a human homologue of rat pttg, is overexpressed in hematopoietic neoplasms. Evidence for a transcriptional activation function of hPTTG. Oncogene 1998;17(17):2187-93.
26. Pei L. Identification of c-myc as a down-stream target for pituitary tumor-transforming gene. J Biol Chem 2001;276(11):8484-91.
27. Horwitz GA, Miklovsky I, Heaney AP, Ren SG, Melmed S. Human pituitary tumor-transforming gene (PTTG1) motif suppresses prolactin expression. Mol Endocrinol 2003;17(4):600-9.
28. Ishikawa H, Heaney AP, Yu R, Horwitz GA, Melmed S. Human pituitary tumor-transforming gene induces angiogenesis. J Clin Endocrinol Metab 2001;86(2):867-74.
29. McCabe CJ, Boelaert K, Tannahill LA, Heaney AP, Stratford AL, Khaira JS, et al. Vascular endothelial growth factor, its receptor KDR/Flk-1, and pituitary tumor transforming gene in pituitary tumors. J Clin Endocrinol Metab 2002;87(9):4238-44.
30. Heaney AP, Horwitz GA, Wang Z, Singson R, Melmed S. Early involvement of estrogen-induced pituitary tumor transforming gene and fibroblast growth factor expression in prolactinoma pathogenesis. Nat Med 1999;5(11):1317-21.
31. Yin H, Fujimoto N, Maruyama S, Asano K. Strain difference in regulation of pituitary tumor transforming gene (PTTG) in estrogen-induced pituitary tumorigenesis in rats. Jpn J Cancer Res 2001;92(10):1034-40.
32. Chien W, Pei L. A novel binding factor facilitates nuclear translocation and transcriptional activation function of the pituitary tumor-transforming gene product. J Biol Chem 2000;275(25): 19422-7.
33. Pei L. Pituitary tumor-transforming gene protein associates with ribosomal protein S10 and a novel human homologue of DnaJ in testicular cells. J Biol Chem 1999;274(5):3151-8.
34. Bernal JA, Luna R, Espina A, Lazaro I, Ramos-Morales F, Romero F, et al. Human securin interacts with p53 and modulates p53-mediated transcriptional activity and apoptosis. Nat Genet 2002;32(2):306-11.
35. Stratford AL, Boelaert K, Tannahill LA, Kim DS, Warfield A, Eggo MC, et al. Pituitary tumor transforming gene binding factor: a novel transforming gene in thyroid tumorigenesis. J Clin Endocrinol Metab 2005;90(7):4341-9.
36. Smith GC, Divecha N, Lakin ND, Jackson SP. DNA-dependent protein kinase and related proteins. Biochem Soc Symp 1999;64:91-104.
37. Lieber MR. The biochemistry and biological significance of nonhomologous DNA end joining: an essential repair process in multicellular eukaryotes. Genes Cells 1999;4(2):77-85.
38. Kim D, Pemberton H, Stratford AL, Buelaert K, Watkinson JC, Lopes V, et al. Pituitary tumour transforming gene (PTTG) induces genetic instability in thyroid cells. Oncogene 2005;24(30):4861-6.
39. McCabe C. Genetic targets for the treatment of pituitary adenomas: focus on the pituitary tumor transforming gene. Curr Opin Pharmacol 2001; 1 (6):620-5.
40. Pei L. Activation of mitogen-activated protein kinase cascade regulates pituitary tumor-transforming gene transactivation function. J Biol Chem 2000;275(40):31191-8.
41. Boelaert K, Yu R, Tannahill LA, Stratford AL, Khanim FL, Eggo MC, et al. PTTG's C-terminal PXXP motifs modulate critical cellular processes in vitro. J Mol Endocrinol 2004;33(3):663-77.
42. Bellail AC, Hunter SB, Brat DJ, Tan C, Van Meir EG. Microregional extracellular matrix heterogeneity in brain modulates glioma cell invasion. Int J Biochem Cell Biol 2004;36(6): 1046-69.
43. Ramnath N, Creaven PJ. Matrix metalloproteinase inhibitors. Curr Oncol Rep 2004;6(2):96-102.
44. Goldman S, Shalev E. The role of the matrix metalloproteinases in human endometrial and ovarian cycles. Eur J Obstet Gynecol Reprod Biol 2003;l 11(2):109-21.
45. Kadoglou NP, Liapis CD. Matrix metalloproteinases: contribution to pathogenesis, diagnosis, surveillance and treatment of abdominal aortic aneurysms. Curr Med Res Opin 2004;20(4):419-32.
46. Peterson JT. Matrix metalloproteinase inhibitor development and the remodeling of drug discovery. Heart Fail Rev 2004;9(1):63-79.
47. Friedberg MH, Glantz MJ, Klempner MS, Cole BF, Perides G. Specific matrix metalloproteinase profiles in the cerebrospinal fluid correlated with the presence of malignant astrocytomas, brain metastases, and carcinomatous meningitis. Cancer 1998;82(5):923-30.
48. Nakagawa T, Kubota T, Kabuto M, Kodera T. Captopril inhibits glioma cell invasion in vitro: involvement of matrix metalloproteinases. Anticancer Res 1995; 15(5B): 1985-9.
49. Hur JH, Park MJ, Park IC, Yi DH, Rhee CH, Hong SI, et al. Matrix metalloproteinases in human gliomas: activation of matrix metalloproteinase-2 (MMP-2) may be correlated with membrane-type-1 matrix metalloproteinase (MT 1-MMP) expression. J Korean Med Sci 2000;15(3):309-14.
50. Raithatha SA, Muzik H, Muzik H, Rewcastle NB, Johnston RN, Edwards DR, et al. Localization of gelatinase-A and gelatinase-B mRNA and protein in human gliomas. Neuro Oncol 2000;2(3): 145-50.
51. da Rocha AB, Mans DR, Lenz G, Fernandes AK, de Lima C, Monteiro VF, et al. Protein kinase C-mediated in vitro invasion of human glioma cells through extracellular-signal-regulated kinase and ornithine decarboxylase. Pathobiology 2000;68(3):l 13-23.
52. Berezikov E, Guryev V, van de Belt J, Wienholds E, Plasterk RH, Cuppen E. Phylogenetic shadowing and computational identification of human microRNA genes. Cell 2005;120(1):21-4.
53. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005;120(1):15-20.
54. Llave C, Xie Z, Kasschau KD, Carrington JC. Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 2002;297(5589):2053-6.
55. Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E, et al. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13ql4 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 2002;99(24): 15524-9.
56. Hamid T, Malik MT, Kakar SS. Ectopic expression of PTTG1/securin promotes tumorigenesis in human embryonic kidney cells. Mol Cancer 2005;4(1):3.
57. Liu CL, Yu IS, Pan HW, Lin SW, Hsu HC. L2dtl is essential for cell survival and nuclear division in early mouse embryonic development. J Biol Chem 2007;282(2): 1109-18.
58. Yu R, Heaney AP, Lu W, Chen J, Melmed S. Pituitary tumor transforming gene causes aneuploidy and p53-dependent and p53-independent apoptosis. J Biol Chem 2000;275(47):36502-5.
59. Hornig NC, Knowles PP, McDonald NQ, Uhhnann F. The dual mechanism of separase regulation by securin. Curr Biol 2002;12(12):973-82.
60. Solbach C, Roller M, Peters S, Nicoletti M, Kaufmann M, Knecht R. Pituitary tumor-transforming gene (PTTG): a novel target for anti-tumor therapy. Anticancer Res 2005;25(1A):121-5.
61. Boelaert K, Tannahill LA, Bulmer JN, Kachilele S, Chan SY, Kim D, et al. A potential role for PTTG/securin in the developing human fetal brain. FASEB J 2003; 17(12): 1631-9.
62. Yang XH, Sladek TL, Liu X, Butler BR, Froelich CJ, Thor AD. Reconstitution of caspase 3 sensitizes MCF-7 breast cancer cells to doxorubicin- and etoposide-induced apoptosis. Cancer Res 2001;61(1):348-54.
63. Basanez G, Zhang J, Chau BN, Maksaev GI, Frolov VA, Brandt TA, et al. Pro-apoptotic cleavage products of Bcl-xL form cytochrome c-conducting pores in pure lipid membranes. J Biol Chem 2001 ;276(33):31083-91.
64. Kho PS, Wang Z, Zhuang L, Li Y, Chew JL, Ng HH, et al. p53-regulated transcriptional program associated with genotoxic stress-induced apoptosis. J Biol Chem 2004;279(20):21183-92.
65. Zhou Y, Mehta KR, Choi AP, Scolavino S, Zhang X. DNA damage-induced inhibition of securin expression is mediated by p53. J Biol Chem 2003;278(1):462-70.
66. Hamid T, Kakar SS. PTTG/securin activates expression of p53 and modulates its function. Mol Cancer 2004;3:18.
67. Jung CR, Hwang KS, Yoo J, Cho WK, Kim JM, Kim WH, et al. E2-EPF UCP targets pVHL for degradation and associates with tumor growth and metastasis. Nat Med 2006;12(7):809-16.
68. Tong Y, Tan Y, Zhou C, Melmed S. Pituitary tumor transforming gene interacts with Sp1 to modulate G1/S cell phase transition. Oncogene 2007;26(38):5596-605.
69. Chen L, Liu YS, Wang LS, Yin HG, Hou QT, Liu ZX, et al. [Expression of pituitary tumor transforming gene, endostatin, and basic fibroblast growth factor mRNAs in invasive pituitary adenomas]. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2004;29(6):651-3, 666.
70. Bogdanos J, Karamanolakis D, Tenta R, Tsintavis A, Milathianakis C, Mitsiades C, et al. Endocrine/paracrine/autocrine survival factor activity of bone microenvironment participates in the development of androgen ablation and chemotherapy refractoriness of prostate cancer metastasis in skeleton. Endocr Relat Cancer 2003;10(2):279-89.
71. Aguayo A, Giles F, Albitar M. Vascularity, angiogenesis and angiogenic factors in leukemias and myelodysplastic syndromes. Leuk Lymphoma 2003;44(2):213-22.
72. Kim DS, Franklyn JA, Stratford AL, Boelaert K, Watkinson JC, Eggo MC, et al. Pituitary tumor-transforming gene regulates multiple downstream angiogenic genes in thyroid cancer. J Clin Endocrinol Metab 2006;91(3):1119-28.
73. Chen G, Li J, Li F, Li X, Zhou J, Lu Y, et al. Inhibitory effects of anti-sense PTTG on malignant phenotype of human ovarian carcinoma cell line SK-OV-3. J Huazhong Univ Sci Technolog Med Sci 2004;24(4):369-72.
2. Zhang X, Horwitz GA, Prezant TR, Valentini A, Nakashima M, Bronstein MD, et al. Structure, expression, and function of human pituitary tumor-transforming gene (PTTG). Mol Endocrinol 1999;13(1):156-66.
3. Chen L, Puri R, Lefkowitz EJ, Kakar SS. Identification of the human pituitary tumor transforming gene (hPTTG) family: molecular structure, expression, and chromosomal localization. Gene 2000;248(1-2):41-50.
4. Saez C, Japon MA, Ramos-Morales F, Romero F, Segura DI, Tortolero M, et al. hpttg is over-expressed in pituitary adenomas and other primary epithelial neoplasias. Oncogene 1999;18(39):5473-6.
5. Filippella M, Galland F, Kujas M, Young J, Faggiano A, Lombardi G, et al. Pituitary tumour transforming gene (PTTG) expression correlates with the proliferative activity and recurrence status of pituitary adenomas: a clinical and immunohistochemical study. Clin Endocrinol (Oxf) 2006;65(4):536-43.
6. Heaney AP, Nelson V, Fernando M, Horwitz G. Transforming events in thyroid tumorigenesis and their association with follicular lesions. J Clin Endocrinol Metab 2001;86(10):5025-32.
7. Kakar SS, Jennes L. Molecular cloning and characterization of the tumor transforming gene (TUTR1): a novel gene in human tumorigenesis. Cytogenet Cell Genet 1999;84(3-4):211-6.
8. Thompson AD 3rd, Kakar SS. Insulin and IGF-I regulate the expression of the pituitary tumor transforming gene (PTTG) in breast tumor cells. FEBS Lett 2005;579(14):3195-200.
9. Tsai SJ, Lin SJ, Cheng YM, Chen HM, Wing LY. Expression and functional analysis of pituitary tumor transforming gene-1 [corrected] in uterine leiomyomas. J Clin Endocrinol Metab 2005;90(6):3715-23.
10. Kakar SS, Malik MT. Suppression of lung cancer with siRNA targeting PTTG. Int J Oncol 2006;29(2):387-95.
11. Rehfeld N, Geddert H, Atamna A, Rohrbeck A, Garcia G, Kliszewski S, et al. The influence of the pituitary tumor transforming gene-1 (PTTG-1) on survival of patients with small cell lung cancer and non-small cell lung cancer. J Carcinog 2006;5:4.
12. Mu YM, Oba K, Yanase T, Ito T, Ashida K, Goto K, et al. Human pituitary tumor transforming gene (hPTTG) inhibits human lung cancer A549 cell growth through activation of p21(WAF1/CIP1 ). Endocr J 2003;50(6):771-81.
13. Honda S. Hayashi M, Kobayashi Y, Ishikawa Y, Nakagawa K, Tsuchiya E. A role for the pituitary tumor-transforming gene in the genesis and progression of non-small cell lung carcinomas. Anticancer Res 2003;23(5A):3775-82.
14. Akino K, Akita S, Mizuguchi T, Takumi I, Yu R, Wang XY, et al. A novel molecular marker of pituitary tumor transforming gene involves in a rat liver regeneration. J Surg Res 2005;129(1): 142-6.
15. Cho-Rok J, Yoo J, Jang YJ, Kim S, Chu IS, Yeom YI, et al. Adenovirus-mediated transfer of siRNA against PTTGl inhibits liver cancer cell growth in vitro and in vivo. Hepatology 2006;43(5): 1042-52.
16. Zhou C, Liu S, Zhou X, Xue L, Quan L, Lu N, et al. Overexpression of human pituitary tumor transforming gene (hPTTG), is regulated by beta-catenin /TCF pathway in human esophageal squamous cell carcinoma. Int J Cancer 2005; 113(6):891-8.
17. Heaney AP, Singson R, McCabe CJ, Nelson V, Nakashima M, Melmed S. Expression of pituitary-tumour transforming gene in colorectal tumours. Lancet 2000;355(9205):716-9.
18. Chamaon K, Kirches E, Kanakis D, Braeuninger S, Dietzmann K, Mawrin C. Regulation of the pituitary tumor transforming gene by insulin-like-growth factor-I and insulin differs between malignant and non-neoplastic astrocytes. Biochem Biophys Res Commun 2005;331(1):86-92.
19. Tfelt-Hansen J, Yano S, Bandyopadhyay S, Carroll R, Brown EM, Chattopadhyay N. Expression of pituitary tumor transforming gene (PTTG) and its binding protein in human astrocytes and astrocytoma cells: function and regulation of PTTG in U87 astrocytoma cells. Endocrinology 2004;145(9):4222-31.
20. Wang Z, Melmed S. Pituitary tumor transforming gene (PTTG) transforming and transactivation activity. J Biol Chem 2000;275(11):7459-61.
21. Ramos-Morales F, Dominguez A, Romero F, Luna R, Multon MC, Pintor-Toro JA, et al. Cell cycle regulated expression and phosphorylation of hpttg proto-oncogene product. Oncogene 2000;19(3):403-9.
22. Yu R, Ren SG, Horwitz GA, Wang Z, Melmed S. Pituitary tumor transforming gene (PTTG) regulates placental JEG-3 cell division and survival: evidence from live cell imaging. Mol Endocrinol 2000; 14(8): 1137-46.
23. Zou H, McGarry TJ, Bernal T, Kirschner MW. Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis. Science 1999;285(5426):418-22.
24. Romero F, Multon MC, Ramos-Morales F, Dominguez A, Bernal JA, Pintor-Toro JA, et al. Human securin, hPTTG, is associated with Ku heterodimer, the regulatory subunit of the DNA-dependent protein kinase. Nucleic Acids Res 2001 ;29(6): 1300-7.
25. Dominguez A, Ramos-Morales F, Romero F, Rios RM, Dreyfus F, Tortolero M, et al. hpttg, a human homologue of rat pttg, is overexpressed in hematopoietic neoplasms. Evidence for a transcriptional activation function of hPTTG. Oncogene 1998;17(17):2187-93.
26. Pei L. Identification of c-myc as a down-stream target for pituitary tumor-transforming gene. J Biol Chem 2001;276(11):8484-91.
27. Horwitz GA, Miklovsky I, Heaney AP, Ren SG, Melmed S. Human pituitary tumor-transforming gene (PTTG1) motif suppresses prolactin expression. Mol Endocrinol 2003;17(4):600-9.
28. Ishikawa H, Heaney AP, Yu R, Horwitz GA, Melmed S. Human pituitary tumor-transforming gene induces angiogenesis. J Clin Endocrinol Metab 2001;86(2):867-74.
29. McCabe CJ, Boelaert K, Tannahill LA, Heaney AP, Stratford AL, Khaira JS, et al. Vascular endothelial growth factor, its receptor KDR/Flk-1, and pituitary tumor transforming gene in pituitary tumors. J Clin Endocrinol Metab 2002;87(9):4238-44.
30. Heaney AP, Horwitz GA, Wang Z, Singson R, Melmed S. Early involvement of estrogen-induced pituitary tumor transforming gene and fibroblast growth factor expression in prolactinoma pathogenesis. Nat Med 1999;5(11):1317-21.
31. Yin H, Fujimoto N, Maruyama S, Asano K. Strain difference in regulation of pituitary tumor transforming gene (PTTG) in estrogen-induced pituitary tumorigenesis in rats. Jpn J Cancer Res 2001;92(10):1034-40.
32. Chien W, Pei L. A novel binding factor facilitates nuclear translocation and transcriptional activation function of the pituitary tumor-transforming gene product. J Biol Chem 2000;275(25): 19422-7.
33. Pei L. Pituitary tumor-transforming gene protein associates with ribosomal protein S10 and a novel human homologue of DnaJ in testicular cells. J Biol Chem 1999;274(5):3151-8.
34. Bernal JA, Luna R, Espina A, Lazaro I, Ramos-Morales F, Romero F, et al. Human securin interacts with p53 and modulates p53-mediated transcriptional activity and apoptosis. Nat Genet 2002;32(2):306-11.
35. Stratford AL, Boelaert K, Tannahill LA, Kim DS, Warfield A, Eggo MC, et al. Pituitary tumor transforming gene binding factor: a novel transforming gene in thyroid tumorigenesis. J Clin Endocrinol Metab 2005;90(7):4341-9.
36. Smith GC, Divecha N, Lakin ND, Jackson SP. DNA-dependent protein kinase and related proteins. Biochem Soc Symp 1999;64:91-104.
37. Lieber MR. The biochemistry and biological significance of nonhomologous DNA end joining: an essential repair process in multicellular eukaryotes. Genes Cells 1999;4(2):77-85.
38. Kim D, Pemberton H, Stratford AL, Buelaert K, Watkinson JC, Lopes V, et al. Pituitary tumour transforming gene (PTTG) induces genetic instability in thyroid cells. Oncogene 2005;24(30):4861-6.
39. McCabe C. Genetic targets for the treatment of pituitary adenomas: focus on the pituitary tumor transforming gene. Curr Opin Pharmacol 2001; 1 (6):620-5.
40. Pei L. Activation of mitogen-activated protein kinase cascade regulates pituitary tumor-transforming gene transactivation function. J Biol Chem 2000;275(40):31191-8.
41. Boelaert K, Yu R, Tannahill LA, Stratford AL, Khanim FL, Eggo MC, et al. PTTG's C-terminal PXXP motifs modulate critical cellular processes in vitro. J Mol Endocrinol 2004;33(3):663-77.
42. Bellail AC, Hunter SB, Brat DJ, Tan C, Van Meir EG. Microregional extracellular matrix heterogeneity in brain modulates glioma cell invasion. Int J Biochem Cell Biol 2004;36(6): 1046-69.
43. Ramnath N, Creaven PJ. Matrix metalloproteinase inhibitors. Curr Oncol Rep 2004;6(2):96-102.
44. Goldman S, Shalev E. The role of the matrix metalloproteinases in human endometrial and ovarian cycles. Eur J Obstet Gynecol Reprod Biol 2003;l 11(2):109-21.
45. Kadoglou NP, Liapis CD. Matrix metalloproteinases: contribution to pathogenesis, diagnosis, surveillance and treatment of abdominal aortic aneurysms. Curr Med Res Opin 2004;20(4):419-32.
46. Peterson JT. Matrix metalloproteinase inhibitor development and the remodeling of drug discovery. Heart Fail Rev 2004;9(1):63-79.
47. Friedberg MH, Glantz MJ, Klempner MS, Cole BF, Perides G. Specific matrix metalloproteinase profiles in the cerebrospinal fluid correlated with the presence of malignant astrocytomas, brain metastases, and carcinomatous meningitis. Cancer 1998;82(5):923-30.
48. Nakagawa T, Kubota T, Kabuto M, Kodera T. Captopril inhibits glioma cell invasion in vitro: involvement of matrix metalloproteinases. Anticancer Res 1995; 15(5B): 1985-9.
49. Hur JH, Park MJ, Park IC, Yi DH, Rhee CH, Hong SI, et al. Matrix metalloproteinases in human gliomas: activation of matrix metalloproteinase-2 (MMP-2) may be correlated with membrane-type-1 matrix metalloproteinase (MT 1-MMP) expression. J Korean Med Sci 2000;15(3):309-14.
50. Raithatha SA, Muzik H, Muzik H, Rewcastle NB, Johnston RN, Edwards DR, et al. Localization of gelatinase-A and gelatinase-B mRNA and protein in human gliomas. Neuro Oncol 2000;2(3): 145-50.
51. da Rocha AB, Mans DR, Lenz G, Fernandes AK, de Lima C, Monteiro VF, et al. Protein kinase C-mediated in vitro invasion of human glioma cells through extracellular-signal-regulated kinase and ornithine decarboxylase. Pathobiology 2000;68(3):l 13-23.
52. Berezikov E, Guryev V, van de Belt J, Wienholds E, Plasterk RH, Cuppen E. Phylogenetic shadowing and computational identification of human microRNA genes. Cell 2005;120(1):21-4.
53. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005;120(1):15-20.
54. Llave C, Xie Z, Kasschau KD, Carrington JC. Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 2002;297(5589):2053-6.
55. Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E, et al. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13ql4 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 2002;99(24): 15524-9.
56. Hamid T, Malik MT, Kakar SS. Ectopic expression of PTTG1/securin promotes tumorigenesis in human embryonic kidney cells. Mol Cancer 2005;4(1):3.
57. Liu CL, Yu IS, Pan HW, Lin SW, Hsu HC. L2dtl is essential for cell survival and nuclear division in early mouse embryonic development. J Biol Chem 2007;282(2): 1109-18.
58. Yu R, Heaney AP, Lu W, Chen J, Melmed S. Pituitary tumor transforming gene causes aneuploidy and p53-dependent and p53-independent apoptosis. J Biol Chem 2000;275(47):36502-5.
59. Hornig NC, Knowles PP, McDonald NQ, Uhhnann F. The dual mechanism of separase regulation by securin. Curr Biol 2002;12(12):973-82.
60. Solbach C, Roller M, Peters S, Nicoletti M, Kaufmann M, Knecht R. Pituitary tumor-transforming gene (PTTG): a novel target for anti-tumor therapy. Anticancer Res 2005;25(1A):121-5.
61. Boelaert K, Tannahill LA, Bulmer JN, Kachilele S, Chan SY, Kim D, et al. A potential role for PTTG/securin in the developing human fetal brain. FASEB J 2003; 17(12): 1631-9.
62. Yang XH, Sladek TL, Liu X, Butler BR, Froelich CJ, Thor AD. Reconstitution of caspase 3 sensitizes MCF-7 breast cancer cells to doxorubicin- and etoposide-induced apoptosis. Cancer Res 2001;61(1):348-54.
63. Basanez G, Zhang J, Chau BN, Maksaev GI, Frolov VA, Brandt TA, et al. Pro-apoptotic cleavage products of Bcl-xL form cytochrome c-conducting pores in pure lipid membranes. J Biol Chem 2001 ;276(33):31083-91.
64. Kho PS, Wang Z, Zhuang L, Li Y, Chew JL, Ng HH, et al. p53-regulated transcriptional program associated with genotoxic stress-induced apoptosis. J Biol Chem 2004;279(20):21183-92.
65. Zhou Y, Mehta KR, Choi AP, Scolavino S, Zhang X. DNA damage-induced inhibition of securin expression is mediated by p53. J Biol Chem 2003;278(1):462-70.
66. Hamid T, Kakar SS. PTTG/securin activates expression of p53 and modulates its function. Mol Cancer 2004;3:18.
67. Jung CR, Hwang KS, Yoo J, Cho WK, Kim JM, Kim WH, et al. E2-EPF UCP targets pVHL for degradation and associates with tumor growth and metastasis. Nat Med 2006;12(7):809-16.
68. Tong Y, Tan Y, Zhou C, Melmed S. Pituitary tumor transforming gene interacts with Sp1 to modulate G1/S cell phase transition. Oncogene 2007;26(38):5596-605.
69. Chen L, Liu YS, Wang LS, Yin HG, Hou QT, Liu ZX, et al. [Expression of pituitary tumor transforming gene, endostatin, and basic fibroblast growth factor mRNAs in invasive pituitary adenomas]. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2004;29(6):651-3, 666.
70. Bogdanos J, Karamanolakis D, Tenta R, Tsintavis A, Milathianakis C, Mitsiades C, et al. Endocrine/paracrine/autocrine survival factor activity of bone microenvironment participates in the development of androgen ablation and chemotherapy refractoriness of prostate cancer metastasis in skeleton. Endocr Relat Cancer 2003;10(2):279-89.
71. Aguayo A, Giles F, Albitar M. Vascularity, angiogenesis and angiogenic factors in leukemias and myelodysplastic syndromes. Leuk Lymphoma 2003;44(2):213-22.
72. Kim DS, Franklyn JA, Stratford AL, Boelaert K, Watkinson JC, Eggo MC, et al. Pituitary tumor-transforming gene regulates multiple downstream angiogenic genes in thyroid cancer. J Clin Endocrinol Metab 2006;91(3):1119-28.
73. Chen G, Li J, Li F, Li X, Zhou J, Lu Y, et al. Inhibitory effects of anti-sense PTTG on malignant phenotype of human ovarian carcinoma cell line SK-OV-3. J Huazhong Univ Sci Technolog Med Sci 2004;24(4):369-72.