NEDD4-1、PTEN在垂体瘤中的表达及作用的研究
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摘要
第一部分：
NEDD4-1、PTEN在垂体瘤中的表达及相关性的研究
研究背景
垂体腺瘤是临床内分泌科和神经科的常见病,是颅内常见的良性肿瘤,其发病率排在脑胶质瘤和脑膜瘤之后,居颅内肿瘤发病率的第三位。垂体腺瘤在组织学上属于良性肿瘤,但也有一些垂体瘤具有向周围组织浸润性生长的特点,称为侵袭性垂体瘤。侵袭性垂体瘤虽在病理上属于良性肿瘤,但具有向周围正常结构如颅骨、海绵窦、硬脑膜、蝶窦、三脑室、鞍上、鞍旁、斜坡甚至鼻咽腔等位置呈侵袭性生长的特点。目前采取的各种治疗方案均能影响垂体的正常分泌功能,甚至引起低垂体功能,患者长期进行激素替代治疗,痛苦不堪。对垂体瘤的发病机制的研究最近有很大的进展,但确切的机制和机理尚不清楚,因此进一步广泛的研究垂体肿瘤的发生,发展机制将继续成为研究的热点。目前,研究抑癌基因在肿瘤中的变异和表达已成为探索肿瘤病因、发展及预后的一条重要途径,并期望在其中寻找基因治疗的目标基因。发现和阐明相关基因的作用机理,不仅有助于发现能反应肿瘤特异性生物学行为的标志物用于临床诊断和随访研究,更重要的是为制定该类型肿瘤的新型治疗方案提供理论学基础。PTEN(phosphatase and tensin homologue deleted on chromosome10,第10号染色体同源丢失性磷酸酶-张力蛋白基因)是1997年由三个研究小组(Li& Sun1997,Li etal.1997, Steck et al.1997)同时克隆并命名的一种抑癌基因。PTEN定位于10q23.3,由9个外显子组成,编码由403个氨基酸组成的在细胞质中表现出双重特异性磷酸酶活性的蛋白,PTEN是迄今发现的第一个具有磷酸酶活性的肿瘤抑制基因。国内外大量研究证实,PTEN与脑胶质瘤、子宫内膜癌、乳腺癌、前列腺癌、肝癌的发生、发展有密切关系,而且肿瘤的恶性程度愈高,PTEN表达率愈低,但目前关于垂体瘤中PTEN基因的缺失国内外报道较少。近十余年来,研究已发现PTEN可通过抑制血管生成、阻抑细胞周期、抑制细胞外基质降解、促进细胞分化等作用抑制肿瘤生成。但对肿瘤中PTEN的调控和降解机制的研究较少。NEDD4家族为一类具有E3泛素连接酶活性的蛋白质,NEDD4家族在进化上高度保守,具有一个C2(Ca2+/lipid-binding)结构域,2-4个WW结构域以及1个HECT (homologous to the E6-AP carboxyl terminus)结构域。C2参与膜靶向结合,WW参与蛋白质的相互作用以及底物识别,HECT具有E3的催化活性,负责靶蛋白的泛素化和降解。2007年,Xuejun Jiang实验室鉴定出一种调节PTEN的新成分——NEDD4-1(neural precursor cell expressed developmentally down regulated4-1, NEDD4-1), NEDD4-1在下调肿瘤抑制基因PTEN水平的过程中发挥重要作用,通过向PTEN附加泛素这一分子标志物,促使PTEN在蛋白酶体中降解。然而,在垂体瘤中是否存在NEDD4-1,其表达情况与垂体瘤侵袭性的关系以及和PTEN的相关性,国内尚未见文献报道。
目的本实验研究NEDD4-1、PTEN分别在正常垂体组织、非侵袭性及侵袭性垂体瘤中的表达，并对其表达与垂体瘤侵袭性程度的关系进行研究,从而为垂体瘤的早期诊断和治疗提供新的生物学特性的参考指标和理论支持。
方法本实验分为正常垂体组织组、非侵袭性垂体瘤组和侵袭性垂体瘤组,正常垂体组织来自术中所取ACTH微腺瘤周围薄层垂体组织。应用RT-PCR法检测40例垂体瘤标本和10例正常垂体组织中的NEDD4-1和PTEN mRNA水平,并进行统计学分析；应用免疫组织化学方法检测10例正常垂体组织标本和50例垂体瘤组织标本中NEDD4-1和PTEN蛋白表达情况,并分析二者之间的相关性。
结果1. RT-PCR方法显示NEDD4-1mRNA和PTEN mRNA在垂体瘤和正常垂体组织均有表达,但NEDD4-1mRNA在垂体瘤中表达明显高于正常垂体组织(P<0.05),在侵袭性垂体瘤组中的表达明显高于非侵袭性垂体瘤组(P<0.05)。而PTEN mRNA在垂体瘤中的表达低于正常垂体组织(P<0.05),且在非侵袭性垂体瘤组和侵袭性垂体瘤组中的表达情况与NEDD4-1相反。
2.免疫组化结果显示,NEDD4-1蛋白免疫组化阳性表达定位于细胞浆和(或)细胞核,NEDD4-1蛋白在正常对照组和实验组中的阳性表达率分别为10.0%(1/10)和52.0%(26/50),二者相比差异有显著性(P<0.05)。NEDD4-1蛋白表达阳性率在非侵袭性垂体瘤组中为37.5%(9/24),在侵袭性垂体瘤组中为65.3%(17/26). NEDD4-1蛋白表达阳性率随肿瘤侵袭性程度增高有明显增高的趋势,侵袭性分组之间有显著差异(P<0.05)与对照组之间有显著差异(P<0.05)。PTEN阳性表达主要定位于细胞浆,呈棕黄色染色。PTEN蛋白在正常对照组和实验组中的阳性表达率分别为100.0%(10/10)和54.0%(27/50),二者相比差异有显著性(P<0.05)。PTEN蛋白表达阳性率在非侵袭性垂体瘤组中为83.3%(20/24),在侵袭性垂体瘤组中为26.9%(7/26)。PTEN蛋白表达阳性率随肿瘤侵袭性增高呈降低的趋势。侵袭性分组之间有显著差异(P<0.05)与对照组之间有显著差异(P<0.05)。在26例NEDD4-1表达阳性的垂体瘤中PTEN阳性表达有4例,而在PTEN阳性表达的27例中NEDD4-1阴性表达有23例,经统计分析二者表达呈负相关(Υ=-0.806,P<0.05)。
结论1.正常垂体组织和侵袭性、非侵袭性垂体瘤中均有NEDD4-1和PTEN表达,并与垂体瘤的侵袭性程度相关。NEDD4-1、PTEN可能与垂体瘤的发生、发展有关。
2.垂体瘤中NEDD4-1和PTEN的表达呈负相关。NEDD4-1、PTEN的同时检测可能对判断垂体瘤的生物学特性及预后提供参考。
第二部分：
NEDD4-1对人垂体瘤细胞中PTEN作用的实验研究
目的本实验第一部分研究表明,NEDD4-1与PTEN在垂体瘤的发生、发展过程中可能起着重要的作用,与垂体瘤的侵袭性程度和预后有关,从而为垂体瘤的早期诊断和治疗提供新的生物学特性的参考指标和理论支持。但是NEDD4-1与PTEN在垂体瘤中具体的作用机制还不是很清楚,尚有待进一步的试验研究。本实验第二部分进一步探讨NEDD4-1对GT1.1人垂体瘤细胞中PTEN的作用,为垂体瘤的治疗提供新的途径。
方法构建NEDD4-1shRNA表达质粒,酶切、测序鉴定后扩增。然后脂质体2000分别转染pcDNA3.1-NEDD4-1、pGPU6/GFP/Neo-NEDD4-1shRNA及阴性质粒到GT1.1人垂体瘤细胞,经荧光显微镜观察shRNA质粒转染效率确定最佳时间点。分别采用Western Blot、RT-PCR于转染后第48h检测PTEN的蛋白含量及mRNA水平变化。
结果1. NEDD4-1shRNA表达质粒的鉴定：将重组NEDD4-1shRNA表达质粒转化感受态细胞DH5a,提取的质粒用BamHⅠ和PstⅠ内切酶分别单酶切,结果显示有2条带,1条为超螺旋的SC构型,1条为质粒的松弛开环的OC构型,可显示重组体不能被PstⅠ所酶切。重组体被BamHⅠ单酶切而线性化,条带大小为5100bp。送目的质粒DNA去上海生工生物技术公司完成测序,T3启动子引物5'-AATTA ACCCTCACTAAAGGG-3,结果显示插入序列完全与设计的shRNA碱基序列完全一致,无突变。2.pcDNA3.1-NEDD4-1真核表达质粒的鉴定：将获赠的pcDNA3.1(+)表达载体转化感受态宿主细胞DH5a,提取的质粒经Hind Ⅲ/EcoRI双酶切,1.5%琼脂糖凝胶电泳鉴定重组子,显示2条DNA片段,一条与pcDNA3.1碱基数相符,约为5.4kb,另一条DNA片段(约5.6Kb)与NEDD4-1碱基数一致,证明获赠质粒为NEDD4-1与pcDNA3.1形成的重组体。3.倒置荧光显微镜下观察转染效率：分别于转染后24h、48h、72h在倒置荧光显微镜下观察转染效率,结果显示：转染组可见绿色荧光,而未转染细胞组未见荧光,提示转染成功。转染后24h,即见到绿色荧光,转染后48h左右表达最强,而72h后绿色荧光减弱、细胞大量死亡。故本实验选择在48h检测PTEN mRNA水平及蛋白表达。4.RT-PCR检测四组细胞PTEN基因表达：RT-PCR产物凝胶电泳结果显示：各组相比条带亮度均无明显变化。利用ImageJ分析软件计算出平均灰度值,将其转化为光密度(OD)后进行定量分析,证明：转染NEDD4-1或NEDD4-1shRNA均不能引起GT1.1人垂体瘤细胞中PTEN基因表达的变化。5. Western Blot检测PTEN蛋白表达：显色后扫描成像显示：与对照组相比,NEDD4-1组条带灰度下降,而NEDD4-1干扰组灰度增加,阴性质粒组条带无明显变化。结果提示：(1)转染NEDD4-1后下调了GT1.1人垂体瘤细胞PTEN蛋白表达。(2)转染NEDD4-1shRNA后上调了GT1.1人垂体瘤细胞PTEN蛋白表达。本实验成功构建NEDD4-1的shRNA表达质粒。经稳定转染后,与空白对照组相比,NEDD4-1组PTEN蛋白含量明显减少(P<0.05),而PTEN mRNA变化不明显(P>0.05),NEDD4-1干扰组PTEN蛋白含量明显增加(P<0.05),而PTENmRNA变化不明显(P>0.05)。
结论垂体瘤细胞中NEDD4-1的异常高表达可通过翻译后过程阻抑PTEN表达。
Part one:
The study of the expression and correlation of NEDD4-1and PTEN in pituitary adenomas
Background
Pituitary adenoma is a kind of common disease in clinical endocrinology and neurology, as well as a common intracranial benign tumor, whose incidence is second only to gliomas and meningiomas and listed in the third place among the intracranial tumors. Pituitary adenomas are histologically classified into benign tumors, but some of them are invasive and characterized by invasive growth towards the surrounding normal structures such as the skull, cavernous sinus, dura, sphenoid, the third ventricle, upper and next to the saddle, the slope and even pharyngonasal cavity. In recent years, a large number of advances in studies on the pathogenesis of pituitary adenoma have been achieved, but the exact mechanism and its cause are still unclear. Therefore, further extensive studies on pituitary adenomas and the occurrence and development mechanisms of invasiveness will be still hot spots. At present, the study on variation and expression of tumor suppressor genes has become an important way in exploring tumor etiology, development and prognosis. It is expected to look for the targeted gene among the tumor suppressor genes for gene therapy. Discovering and elucidating the action mechanism of related genes is not only helpful in finding markers that can reflect the specific tumor biological behaviors for clinical diagnosis and follow-up study, but also can provide theory basis for developing the new therapy of this kind of tumor. PTEN (phosphatase and tensin homologue deleted on chromosome10) is a kind of tumor-suppressor gene, which was simultaneously cloned and named by3research groups in1997(Li& Sun1997, Li et al.1997, Steck et al.1997). PTEN located in10q23.3is made up of9exons. It encodes the protein consisting403amino acids that showed dual specific phosphatase activity. PTEN is the first tumor suppressor gene ever found with phosphatase activity. A large number of domestic and foreign studies have confirmed that PTEN had close relationship with occurrence and development of glioma, endometrial cancer, breast cancer, prostate cancer, liver cancer, and the higher the degree of malignant tumor was, the lower the expression rate of PTEN was. But at the moment there were fewer reports about the PTEN gene deletion in the pituitary. For the latest10years, studies have found that PTEN can inhibit tumor formation through inhibiting angiogenesis, obstructing the cell cycle, inhibiting extracellular matrix (ECM) degradation, and promoting cell differentiation, etc. But researches on regulation and the degradation mechanism of PTEN are few. NEDD4family is the kind of protein with E3ubiquitin ligase activity. NEDD4family is highly conserved in evolution, with a C2(Ca2+/lipid-binding) domain,2～4WW domain and1HECT (homologous to the E6-AP carboxyl terminus) domain. C2is involved in membrane targeting, WW participates in protein interaction and the substrate recognition, and HECT with catalytic activity of E3is responsible for the ubiquitination and degradation of target protein. In2007, a new component regulating PTEN, neural precursor cell expressed developmentally down regulated4-1(NEDD4-1) was identified by Xuejun Jiang laboratory. NEDD4-1plays an important role in down-regulation of the level of PTEN, a tumor suppressor gene. It can promote the degradation of PTEN in the proteasome by adding a molecular marker ubiquitin to PTEN. There have neither reports on whether NEDD4-1is existed in pituitary adenomas, nor the relationship of NEDD4-1expression with the invasiveness of pituitary adenomas or with PTEN.
Objective In this study, the expressions of NEDD4-1and PTEN in invasive and non-invasive pituitary adenomas and normal pituitary tissues are analyzed, as well as the relevance between them by using immunohistochemistry. The present study intends to investigate the possible molecular mechanisms that might exist in the occurrence and development of pituitary adenomas, and thereafter to look for the reference indexes for early determination of biological characteristics of pituitary adenomas.
Methods The expression of NEDD4-1mRNA and PTEN mRNA was detected in40pituitary adenomas and10normal pituitary tissuses by using RT-PCR semi-quantity method; immunohistochemistry was used to examine the expression of NEDD4-1and PTEN protein in50pituitary adenomas and10normal pituitary tissuses.
Results1.The expression of NEDD4-1mRNA and PTEN mRNA have been detected in pituitary adenomas and normal pituitary tissuses and the expression level of NEDD4-1mRNA in pituitary adenomas was considerably higher than those in normal pituitary tissuses(P<0.05); the expression level of NEDD4-1mRNA in noninvasive pituitary adenomas and invasive pituitary adenomas was considerably higher than those in normal pituitary tissuses(P<0.05),and the expression level of NEDD4-1mRNA and NEDD4-1protein in invasive pituitary adenomas was considerably higher than those in noninvasive pituitary adenomas.The expression level of PTEN mRNA in pituitary adenomas and in normal pituitary tissuses was opposite to the expression level of NEDD4-1.
2. NEDD4-1protein was positively expressed in the cytoplasm and (or) the nucleus by immunohistochemistry. The positive rates of NEDD4-1protein expressed in the normal control group and the experimental group were10.0%(1/10) and52.0%(26/50), respectively; the difference was significant between them (P<0.05). The positive rate of NEDD4-1protein was37.5%(9/24) in the non-invasive pituitary adenomas group and65.3%(17/26) in the invasive adenomas group. The positive rate of NEDD4-1protein had a significantly increasing trend with the increasing degree of tumor invasiveness, with significant differences between the histological groups (P<0.05); the differences were also significantly compared with the control group (P<0.05). PTEN protein was mainly positively expressed in the cytoplasm, showing brownish yellow. The positive rates of PTEN protein expressed in the normal control group and the experimental group were100.0%(10/10) and54.0%(27/50), respectively; the difference was significant between them (P<0.05). The positive rate of PTEN protein was83.3%(20/24) in the non-invasive pituitary adenomas group and26.9%(7/26) in the invasive pituitary adenomas group. The positive rate of PTEN protein had a significantly increasing trend with the increasing degree of tumor invasiveness, with significant differences between the histological groups (P<0.05); the differences were also significantly compared with the control group (P<0.05). Among26cases of patients with NEDD4-1-positive expression in pituitary adenomas, there were four cased of PTEN-positive expression; among27cases of patients with PTEN-positive expression in pituitary adenomas, there were24cases of NEDD4-1-negative expression. Upon statistical analysis, the expression of both showed a negative correlation (y=-0.806, P<0.05).
Conclusion1. The expression of NEDD4-1and PTEN have been detected in pituitary adenomas and normal pituitary tissuses. The expression of NEDD4-1in pituitary adenomas was considerably high and PTEN was low. The expression of NEDD4-1and PTEN may be correlation to the invasive capacity of pituitary adenomas. NEDD4-1and PTEN may be invoived in the tumorigenesis and progression of pituitary adenomas.
2. There was a strongly negative correlation between NEDD4-1and PTEN, which showed that the high expression of NEDD4-1and nutation of PTEN weren'two isolated events. There might be some correlation between them. Combined inspection of them can be objective index for diagnosis and prognostic in pituitary adenomas in clinical practice. NEDD4-1can be a useful index for diagnosis and prognostic in pituitary adenomas. Also it has a great potential to be a therapeutic target in pituitary adenomas.
Part two:
The study of the effection of NEDD4-1to PTEN in pituitary cells
Objective In the first part of the study showed that, NEDD4-1and PTEN may play an important role in pituitary tumor occurrence and development process, so affected the aggressiveness and prognosis of pituitary tumor, thus providing new theory for the early diagnosis and treatment of pituitary tumors. But the specific mechanism of NEDD4-1and PTEN acted in pituitary adenomas is not clear, further tests have to be studied. In the second part, we study the effection of NEDD4-1to PTEN in GT1.1pituitary cells,therefore a theoretical basis can be provided for pituitary therapy.
Methods Construced the NEDD4-1shRNA-expressed plasmid,then enzyme cutting, sequencing and amplificating. Liposomes2000with pcDNA3.1-NEDD4-l, pGPU6/GFP/Neo-NEDD4-1shRNA and negative plasmid were transfected to the GT1.1pituitary cells. We determined the optimal efficiency of transfection time points by fluorescence microscopy is48hours after transfection. Then we used Western Blot and RT-PCR to detect the protein content and mRNA level of PTEN.
Results1. Identification of NEDD4-1shRNA expression plasmid:Recombined NEDD4-1shRNA expression plasmid was transformed into DH5a competent cells, the extracted plasmid were digested with BamH I or Pst I endonuclease, respectively. The results showed2strips, one was SC (conformation of supercoiled), and the other one was OC (conformation of open loop), suggesting that recombinant cannot be digested by Pst I. Recombinant was digested and linearized by BamH I, with the stripe size of5100bp. Complete sequencing of targeted plasmid DNA was finished in Shanghai Sangon Biotech. T3promoter primer was5'-AATTA ACCCTCACTAAAGGG-3. The results showed that the insertion sequences were completely consistent with the designed shRNA base sequence, and no mutation was found.2. Identification of pcDNA3.1-NEDD4-1eukaryotic expression plasmid:Received pcDNA3.1(+) expression vector was transformed into DH5a competent host cells. The extracted plasmid was digested with Hind Ⅲ/EcoR Ⅰ. With1.5%agarose gel electrophoresis to indentify recombinant, the results showed2strands of DNA fragments:one was consistent with pcDNA3.1base number (about5.4KB), while the other DNA fragments (about5.6KB) was consistent with the NEDD4-1base number. This suggested that the received plasmid was the recombinant formed by NEDD4-1and pcDNA3.1.3. Transfection efficiency was observed under the inverted fluorescence microscope at24h,48h, and72h after transfection, respectively. The result showed that green fluorescent could be seen in transfection group, while not seen in non-transfection group, promoting successful transfection. The green fluorescent was visible at24h after transfection, and reached the strongest intensity at48h. After72h, it became weakened and a large number of cells died. Therefore,48h was selected in this experiment for detection of PTEN mRNA and protein expression.4. RT-PCR detection on PTEN gene expressions in four groups of cells:Gel electrophoresis results of RT-PCR products showed no obvious changes of the stripe brightness compared with each group. Mean grey value was calculated with ImageJ, and transformed into optical density (OD) for quantitative analysis. It was proved that neither transfected NEDD4-1nor NEDD4-1shRNA would result in changes of PTEN gene expression in human pituitary adenoma cells GT1.1.5. PTEN protein expression was detected by Western Blot. Developed scanning imaging showed that compared with the control group, gray level dropped in NEDD4-1group, but was increased in NEDD4-1intervention group. The stripe was not changed obviously in negative plasmid group. Results indicated that:(1) transfected NEDD4-1down-regulated the expression of PTEN protein in pituitary tumor cells GT1.1;(2) transfected NEDD4-1shRNA up-regulated the expression of PTEN protein in pituitary tumor cells GT1.l.NEDD4-1shRNA plasmids were successfully constructed.After the stabilized transfection, compared with the control group,the protein level of PTEN was significantly reduced in NEDD4-1group(P<0.05), but the mRNA level of PTEN did not change significantly (P>0.05), the protein content of PTEN increased significantly in NEDD4-1interference group (P<0.05), while PTENmRNA did not change significantly (P>0.05).
Conclusion The unusually high expression of NEDD4-1in pituitary cells can inhibit PTEN expression by post-translation process.
NEDD4-1、PTEN在垂体瘤中的表达及相关性的研究
研究背景
垂体腺瘤是临床内分泌科和神经科的常见病,是颅内常见的良性肿瘤,其发病率排在脑胶质瘤和脑膜瘤之后,居颅内肿瘤发病率的第三位。垂体腺瘤在组织学上属于良性肿瘤,但也有一些垂体瘤具有向周围组织浸润性生长的特点,称为侵袭性垂体瘤。侵袭性垂体瘤虽在病理上属于良性肿瘤,但具有向周围正常结构如颅骨、海绵窦、硬脑膜、蝶窦、三脑室、鞍上、鞍旁、斜坡甚至鼻咽腔等位置呈侵袭性生长的特点。目前采取的各种治疗方案均能影响垂体的正常分泌功能,甚至引起低垂体功能,患者长期进行激素替代治疗,痛苦不堪。对垂体瘤的发病机制的研究最近有很大的进展,但确切的机制和机理尚不清楚,因此进一步广泛的研究垂体肿瘤的发生,发展机制将继续成为研究的热点。目前,研究抑癌基因在肿瘤中的变异和表达已成为探索肿瘤病因、发展及预后的一条重要途径,并期望在其中寻找基因治疗的目标基因。发现和阐明相关基因的作用机理,不仅有助于发现能反应肿瘤特异性生物学行为的标志物用于临床诊断和随访研究,更重要的是为制定该类型肿瘤的新型治疗方案提供理论学基础。PTEN(phosphatase and tensin homologue deleted on chromosome10,第10号染色体同源丢失性磷酸酶-张力蛋白基因)是1997年由三个研究小组(Li& Sun1997,Li etal.1997, Steck et al.1997)同时克隆并命名的一种抑癌基因。PTEN定位于10q23.3,由9个外显子组成,编码由403个氨基酸组成的在细胞质中表现出双重特异性磷酸酶活性的蛋白,PTEN是迄今发现的第一个具有磷酸酶活性的肿瘤抑制基因。国内外大量研究证实,PTEN与脑胶质瘤、子宫内膜癌、乳腺癌、前列腺癌、肝癌的发生、发展有密切关系,而且肿瘤的恶性程度愈高,PTEN表达率愈低,但目前关于垂体瘤中PTEN基因的缺失国内外报道较少。近十余年来,研究已发现PTEN可通过抑制血管生成、阻抑细胞周期、抑制细胞外基质降解、促进细胞分化等作用抑制肿瘤生成。但对肿瘤中PTEN的调控和降解机制的研究较少。NEDD4家族为一类具有E3泛素连接酶活性的蛋白质,NEDD4家族在进化上高度保守,具有一个C2(Ca2+/lipid-binding)结构域,2-4个WW结构域以及1个HECT (homologous to the E6-AP carboxyl terminus)结构域。C2参与膜靶向结合,WW参与蛋白质的相互作用以及底物识别,HECT具有E3的催化活性,负责靶蛋白的泛素化和降解。2007年,Xuejun Jiang实验室鉴定出一种调节PTEN的新成分——NEDD4-1(neural precursor cell expressed developmentally down regulated4-1, NEDD4-1), NEDD4-1在下调肿瘤抑制基因PTEN水平的过程中发挥重要作用,通过向PTEN附加泛素这一分子标志物,促使PTEN在蛋白酶体中降解。然而,在垂体瘤中是否存在NEDD4-1,其表达情况与垂体瘤侵袭性的关系以及和PTEN的相关性,国内尚未见文献报道。
目的本实验研究NEDD4-1、PTEN分别在正常垂体组织、非侵袭性及侵袭性垂体瘤中的表达，并对其表达与垂体瘤侵袭性程度的关系进行研究,从而为垂体瘤的早期诊断和治疗提供新的生物学特性的参考指标和理论支持。
方法本实验分为正常垂体组织组、非侵袭性垂体瘤组和侵袭性垂体瘤组,正常垂体组织来自术中所取ACTH微腺瘤周围薄层垂体组织。应用RT-PCR法检测40例垂体瘤标本和10例正常垂体组织中的NEDD4-1和PTEN mRNA水平,并进行统计学分析；应用免疫组织化学方法检测10例正常垂体组织标本和50例垂体瘤组织标本中NEDD4-1和PTEN蛋白表达情况,并分析二者之间的相关性。
结果1. RT-PCR方法显示NEDD4-1mRNA和PTEN mRNA在垂体瘤和正常垂体组织均有表达,但NEDD4-1mRNA在垂体瘤中表达明显高于正常垂体组织(P<0.05),在侵袭性垂体瘤组中的表达明显高于非侵袭性垂体瘤组(P<0.05)。而PTEN mRNA在垂体瘤中的表达低于正常垂体组织(P<0.05),且在非侵袭性垂体瘤组和侵袭性垂体瘤组中的表达情况与NEDD4-1相反。
2.免疫组化结果显示,NEDD4-1蛋白免疫组化阳性表达定位于细胞浆和(或)细胞核,NEDD4-1蛋白在正常对照组和实验组中的阳性表达率分别为10.0%(1/10)和52.0%(26/50),二者相比差异有显著性(P<0.05)。NEDD4-1蛋白表达阳性率在非侵袭性垂体瘤组中为37.5%(9/24),在侵袭性垂体瘤组中为65.3%(17/26). NEDD4-1蛋白表达阳性率随肿瘤侵袭性程度增高有明显增高的趋势,侵袭性分组之间有显著差异(P<0.05)与对照组之间有显著差异(P<0.05)。PTEN阳性表达主要定位于细胞浆,呈棕黄色染色。PTEN蛋白在正常对照组和实验组中的阳性表达率分别为100.0%(10/10)和54.0%(27/50),二者相比差异有显著性(P<0.05)。PTEN蛋白表达阳性率在非侵袭性垂体瘤组中为83.3%(20/24),在侵袭性垂体瘤组中为26.9%(7/26)。PTEN蛋白表达阳性率随肿瘤侵袭性增高呈降低的趋势。侵袭性分组之间有显著差异(P<0.05)与对照组之间有显著差异(P<0.05)。在26例NEDD4-1表达阳性的垂体瘤中PTEN阳性表达有4例,而在PTEN阳性表达的27例中NEDD4-1阴性表达有23例,经统计分析二者表达呈负相关(Υ=-0.806,P<0.05)。
结论1.正常垂体组织和侵袭性、非侵袭性垂体瘤中均有NEDD4-1和PTEN表达,并与垂体瘤的侵袭性程度相关。NEDD4-1、PTEN可能与垂体瘤的发生、发展有关。
2.垂体瘤中NEDD4-1和PTEN的表达呈负相关。NEDD4-1、PTEN的同时检测可能对判断垂体瘤的生物学特性及预后提供参考。
第二部分：
NEDD4-1对人垂体瘤细胞中PTEN作用的实验研究
目的本实验第一部分研究表明,NEDD4-1与PTEN在垂体瘤的发生、发展过程中可能起着重要的作用,与垂体瘤的侵袭性程度和预后有关,从而为垂体瘤的早期诊断和治疗提供新的生物学特性的参考指标和理论支持。但是NEDD4-1与PTEN在垂体瘤中具体的作用机制还不是很清楚,尚有待进一步的试验研究。本实验第二部分进一步探讨NEDD4-1对GT1.1人垂体瘤细胞中PTEN的作用,为垂体瘤的治疗提供新的途径。
方法构建NEDD4-1shRNA表达质粒,酶切、测序鉴定后扩增。然后脂质体2000分别转染pcDNA3.1-NEDD4-1、pGPU6/GFP/Neo-NEDD4-1shRNA及阴性质粒到GT1.1人垂体瘤细胞,经荧光显微镜观察shRNA质粒转染效率确定最佳时间点。分别采用Western Blot、RT-PCR于转染后第48h检测PTEN的蛋白含量及mRNA水平变化。
结果1. NEDD4-1shRNA表达质粒的鉴定：将重组NEDD4-1shRNA表达质粒转化感受态细胞DH5a,提取的质粒用BamHⅠ和PstⅠ内切酶分别单酶切,结果显示有2条带,1条为超螺旋的SC构型,1条为质粒的松弛开环的OC构型,可显示重组体不能被PstⅠ所酶切。重组体被BamHⅠ单酶切而线性化,条带大小为5100bp。送目的质粒DNA去上海生工生物技术公司完成测序,T3启动子引物5'-AATTA ACCCTCACTAAAGGG-3,结果显示插入序列完全与设计的shRNA碱基序列完全一致,无突变。2.pcDNA3.1-NEDD4-1真核表达质粒的鉴定：将获赠的pcDNA3.1(+)表达载体转化感受态宿主细胞DH5a,提取的质粒经Hind Ⅲ/EcoRI双酶切,1.5%琼脂糖凝胶电泳鉴定重组子,显示2条DNA片段,一条与pcDNA3.1碱基数相符,约为5.4kb,另一条DNA片段(约5.6Kb)与NEDD4-1碱基数一致,证明获赠质粒为NEDD4-1与pcDNA3.1形成的重组体。3.倒置荧光显微镜下观察转染效率：分别于转染后24h、48h、72h在倒置荧光显微镜下观察转染效率,结果显示：转染组可见绿色荧光,而未转染细胞组未见荧光,提示转染成功。转染后24h,即见到绿色荧光,转染后48h左右表达最强,而72h后绿色荧光减弱、细胞大量死亡。故本实验选择在48h检测PTEN mRNA水平及蛋白表达。4.RT-PCR检测四组细胞PTEN基因表达：RT-PCR产物凝胶电泳结果显示：各组相比条带亮度均无明显变化。利用ImageJ分析软件计算出平均灰度值,将其转化为光密度(OD)后进行定量分析,证明：转染NEDD4-1或NEDD4-1shRNA均不能引起GT1.1人垂体瘤细胞中PTEN基因表达的变化。5. Western Blot检测PTEN蛋白表达：显色后扫描成像显示：与对照组相比,NEDD4-1组条带灰度下降,而NEDD4-1干扰组灰度增加,阴性质粒组条带无明显变化。结果提示：(1)转染NEDD4-1后下调了GT1.1人垂体瘤细胞PTEN蛋白表达。(2)转染NEDD4-1shRNA后上调了GT1.1人垂体瘤细胞PTEN蛋白表达。本实验成功构建NEDD4-1的shRNA表达质粒。经稳定转染后,与空白对照组相比,NEDD4-1组PTEN蛋白含量明显减少(P<0.05),而PTEN mRNA变化不明显(P>0.05),NEDD4-1干扰组PTEN蛋白含量明显增加(P<0.05),而PTENmRNA变化不明显(P>0.05)。
结论垂体瘤细胞中NEDD4-1的异常高表达可通过翻译后过程阻抑PTEN表达。
Part one:
The study of the expression and correlation of NEDD4-1and PTEN in pituitary adenomas
Background
Pituitary adenoma is a kind of common disease in clinical endocrinology and neurology, as well as a common intracranial benign tumor, whose incidence is second only to gliomas and meningiomas and listed in the third place among the intracranial tumors. Pituitary adenomas are histologically classified into benign tumors, but some of them are invasive and characterized by invasive growth towards the surrounding normal structures such as the skull, cavernous sinus, dura, sphenoid, the third ventricle, upper and next to the saddle, the slope and even pharyngonasal cavity. In recent years, a large number of advances in studies on the pathogenesis of pituitary adenoma have been achieved, but the exact mechanism and its cause are still unclear. Therefore, further extensive studies on pituitary adenomas and the occurrence and development mechanisms of invasiveness will be still hot spots. At present, the study on variation and expression of tumor suppressor genes has become an important way in exploring tumor etiology, development and prognosis. It is expected to look for the targeted gene among the tumor suppressor genes for gene therapy. Discovering and elucidating the action mechanism of related genes is not only helpful in finding markers that can reflect the specific tumor biological behaviors for clinical diagnosis and follow-up study, but also can provide theory basis for developing the new therapy of this kind of tumor. PTEN (phosphatase and tensin homologue deleted on chromosome10) is a kind of tumor-suppressor gene, which was simultaneously cloned and named by3research groups in1997(Li& Sun1997, Li et al.1997, Steck et al.1997). PTEN located in10q23.3is made up of9exons. It encodes the protein consisting403amino acids that showed dual specific phosphatase activity. PTEN is the first tumor suppressor gene ever found with phosphatase activity. A large number of domestic and foreign studies have confirmed that PTEN had close relationship with occurrence and development of glioma, endometrial cancer, breast cancer, prostate cancer, liver cancer, and the higher the degree of malignant tumor was, the lower the expression rate of PTEN was. But at the moment there were fewer reports about the PTEN gene deletion in the pituitary. For the latest10years, studies have found that PTEN can inhibit tumor formation through inhibiting angiogenesis, obstructing the cell cycle, inhibiting extracellular matrix (ECM) degradation, and promoting cell differentiation, etc. But researches on regulation and the degradation mechanism of PTEN are few. NEDD4family is the kind of protein with E3ubiquitin ligase activity. NEDD4family is highly conserved in evolution, with a C2(Ca2+/lipid-binding) domain,2～4WW domain and1HECT (homologous to the E6-AP carboxyl terminus) domain. C2is involved in membrane targeting, WW participates in protein interaction and the substrate recognition, and HECT with catalytic activity of E3is responsible for the ubiquitination and degradation of target protein. In2007, a new component regulating PTEN, neural precursor cell expressed developmentally down regulated4-1(NEDD4-1) was identified by Xuejun Jiang laboratory. NEDD4-1plays an important role in down-regulation of the level of PTEN, a tumor suppressor gene. It can promote the degradation of PTEN in the proteasome by adding a molecular marker ubiquitin to PTEN. There have neither reports on whether NEDD4-1is existed in pituitary adenomas, nor the relationship of NEDD4-1expression with the invasiveness of pituitary adenomas or with PTEN.
Objective In this study, the expressions of NEDD4-1and PTEN in invasive and non-invasive pituitary adenomas and normal pituitary tissues are analyzed, as well as the relevance between them by using immunohistochemistry. The present study intends to investigate the possible molecular mechanisms that might exist in the occurrence and development of pituitary adenomas, and thereafter to look for the reference indexes for early determination of biological characteristics of pituitary adenomas.
Methods The expression of NEDD4-1mRNA and PTEN mRNA was detected in40pituitary adenomas and10normal pituitary tissuses by using RT-PCR semi-quantity method; immunohistochemistry was used to examine the expression of NEDD4-1and PTEN protein in50pituitary adenomas and10normal pituitary tissuses.
Results1.The expression of NEDD4-1mRNA and PTEN mRNA have been detected in pituitary adenomas and normal pituitary tissuses and the expression level of NEDD4-1mRNA in pituitary adenomas was considerably higher than those in normal pituitary tissuses(P<0.05); the expression level of NEDD4-1mRNA in noninvasive pituitary adenomas and invasive pituitary adenomas was considerably higher than those in normal pituitary tissuses(P<0.05),and the expression level of NEDD4-1mRNA and NEDD4-1protein in invasive pituitary adenomas was considerably higher than those in noninvasive pituitary adenomas.The expression level of PTEN mRNA in pituitary adenomas and in normal pituitary tissuses was opposite to the expression level of NEDD4-1.
2. NEDD4-1protein was positively expressed in the cytoplasm and (or) the nucleus by immunohistochemistry. The positive rates of NEDD4-1protein expressed in the normal control group and the experimental group were10.0%(1/10) and52.0%(26/50), respectively; the difference was significant between them (P<0.05). The positive rate of NEDD4-1protein was37.5%(9/24) in the non-invasive pituitary adenomas group and65.3%(17/26) in the invasive adenomas group. The positive rate of NEDD4-1protein had a significantly increasing trend with the increasing degree of tumor invasiveness, with significant differences between the histological groups (P<0.05); the differences were also significantly compared with the control group (P<0.05). PTEN protein was mainly positively expressed in the cytoplasm, showing brownish yellow. The positive rates of PTEN protein expressed in the normal control group and the experimental group were100.0%(10/10) and54.0%(27/50), respectively; the difference was significant between them (P<0.05). The positive rate of PTEN protein was83.3%(20/24) in the non-invasive pituitary adenomas group and26.9%(7/26) in the invasive pituitary adenomas group. The positive rate of PTEN protein had a significantly increasing trend with the increasing degree of tumor invasiveness, with significant differences between the histological groups (P<0.05); the differences were also significantly compared with the control group (P<0.05). Among26cases of patients with NEDD4-1-positive expression in pituitary adenomas, there were four cased of PTEN-positive expression; among27cases of patients with PTEN-positive expression in pituitary adenomas, there were24cases of NEDD4-1-negative expression. Upon statistical analysis, the expression of both showed a negative correlation (y=-0.806, P<0.05).
Conclusion1. The expression of NEDD4-1and PTEN have been detected in pituitary adenomas and normal pituitary tissuses. The expression of NEDD4-1in pituitary adenomas was considerably high and PTEN was low. The expression of NEDD4-1and PTEN may be correlation to the invasive capacity of pituitary adenomas. NEDD4-1and PTEN may be invoived in the tumorigenesis and progression of pituitary adenomas.
2. There was a strongly negative correlation between NEDD4-1and PTEN, which showed that the high expression of NEDD4-1and nutation of PTEN weren'two isolated events. There might be some correlation between them. Combined inspection of them can be objective index for diagnosis and prognostic in pituitary adenomas in clinical practice. NEDD4-1can be a useful index for diagnosis and prognostic in pituitary adenomas. Also it has a great potential to be a therapeutic target in pituitary adenomas.
Part two:
The study of the effection of NEDD4-1to PTEN in pituitary cells
Objective In the first part of the study showed that, NEDD4-1and PTEN may play an important role in pituitary tumor occurrence and development process, so affected the aggressiveness and prognosis of pituitary tumor, thus providing new theory for the early diagnosis and treatment of pituitary tumors. But the specific mechanism of NEDD4-1and PTEN acted in pituitary adenomas is not clear, further tests have to be studied. In the second part, we study the effection of NEDD4-1to PTEN in GT1.1pituitary cells,therefore a theoretical basis can be provided for pituitary therapy.
Methods Construced the NEDD4-1shRNA-expressed plasmid,then enzyme cutting, sequencing and amplificating. Liposomes2000with pcDNA3.1-NEDD4-l, pGPU6/GFP/Neo-NEDD4-1shRNA and negative plasmid were transfected to the GT1.1pituitary cells. We determined the optimal efficiency of transfection time points by fluorescence microscopy is48hours after transfection. Then we used Western Blot and RT-PCR to detect the protein content and mRNA level of PTEN.
Results1. Identification of NEDD4-1shRNA expression plasmid:Recombined NEDD4-1shRNA expression plasmid was transformed into DH5a competent cells, the extracted plasmid were digested with BamH I or Pst I endonuclease, respectively. The results showed2strips, one was SC (conformation of supercoiled), and the other one was OC (conformation of open loop), suggesting that recombinant cannot be digested by Pst I. Recombinant was digested and linearized by BamH I, with the stripe size of5100bp. Complete sequencing of targeted plasmid DNA was finished in Shanghai Sangon Biotech. T3promoter primer was5'-AATTA ACCCTCACTAAAGGG-3. The results showed that the insertion sequences were completely consistent with the designed shRNA base sequence, and no mutation was found.2. Identification of pcDNA3.1-NEDD4-1eukaryotic expression plasmid:Received pcDNA3.1(+) expression vector was transformed into DH5a competent host cells. The extracted plasmid was digested with Hind Ⅲ/EcoR Ⅰ. With1.5%agarose gel electrophoresis to indentify recombinant, the results showed2strands of DNA fragments:one was consistent with pcDNA3.1base number (about5.4KB), while the other DNA fragments (about5.6KB) was consistent with the NEDD4-1base number. This suggested that the received plasmid was the recombinant formed by NEDD4-1and pcDNA3.1.3. Transfection efficiency was observed under the inverted fluorescence microscope at24h,48h, and72h after transfection, respectively. The result showed that green fluorescent could be seen in transfection group, while not seen in non-transfection group, promoting successful transfection. The green fluorescent was visible at24h after transfection, and reached the strongest intensity at48h. After72h, it became weakened and a large number of cells died. Therefore,48h was selected in this experiment for detection of PTEN mRNA and protein expression.4. RT-PCR detection on PTEN gene expressions in four groups of cells:Gel electrophoresis results of RT-PCR products showed no obvious changes of the stripe brightness compared with each group. Mean grey value was calculated with ImageJ, and transformed into optical density (OD) for quantitative analysis. It was proved that neither transfected NEDD4-1nor NEDD4-1shRNA would result in changes of PTEN gene expression in human pituitary adenoma cells GT1.1.5. PTEN protein expression was detected by Western Blot. Developed scanning imaging showed that compared with the control group, gray level dropped in NEDD4-1group, but was increased in NEDD4-1intervention group. The stripe was not changed obviously in negative plasmid group. Results indicated that:(1) transfected NEDD4-1down-regulated the expression of PTEN protein in pituitary tumor cells GT1.1;(2) transfected NEDD4-1shRNA up-regulated the expression of PTEN protein in pituitary tumor cells GT1.l.NEDD4-1shRNA plasmids were successfully constructed.After the stabilized transfection, compared with the control group,the protein level of PTEN was significantly reduced in NEDD4-1group(P<0.05), but the mRNA level of PTEN did not change significantly (P>0.05), the protein content of PTEN increased significantly in NEDD4-1interference group (P<0.05), while PTENmRNA did not change significantly (P>0.05).
Conclusion The unusually high expression of NEDD4-1in pituitary cells can inhibit PTEN expression by post-translation process.
引文
1.王忠诚.王忠诚神经外科学武汉,湖北科学技术出版社2005：497-537
2. Palumbo T, Faucz FR, Azevedo M, Xekouki P, Iliopoulos D, Stratakis CA: Functional screen analysis reveals miR-26b and miR-128 as central regulators of pituitary somatomammotrophic tumor growth through activation of the PTEN-AKT pathway.Oncogene.2013 Mar 28;32(13):1651-9
3. Wang X, Trotman L C, Koppie T, et al. NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN.[J]. Cell.2007,128(1):129-139.
4. Wu D N, Pei D S, Wang Q, et al. Down-regulation of PTEN by sodium orthovanadate inhibits ASK1 activation via PI3-K/AKT during cerebral ischemia in rat hippocampus.[J]. Neurosci Lett.2006,404(1-2):98-102.
5. Castellino RC, Durden DL.Mechanisms of Disease:the PI3K-AKT-PTEN signaling node--an intercept point for the control of angiogenesis in brain tumors.Nat Clin Pract Neurol.2007 Dec;3(12):682-93.
6. Tena-Suck ML, Ortiz-Plata A, de la Vega HA. Phosphatase and tensin homologue and pituitary tumor-transforming gene in pituitary adenomas. Clinical-pathologic and immunohistochemical analysis.Ann Diagn Pathol.2008 Aug;12(4):275-82.
7. Feng ZZ, Chen JW, Yang ZR, Lu GZ, Cai ZG Expression of PTTG1 and PTEN in endometrial carcinoma:correlation with tumorigenesis and progression.Med Oncol. 2012 Mar;29(1):304-10
8. Vivanco I & Sawyers CL 2002 The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nature Review Cancer 2 489-501.
9. Whiteman DC, Zhou XP, Cummings MC, Pavey S, Hayward NK & Eng C 2002Nuclear PTEN expression and clinicopathologic features in a population-based series of primary cutaneous melanoma. International Journal of Cancer 9963-67.
10. Faisal Ahmed S, Marsh DJ, Weremowicz S, Morton CC, Williams DM, Eng C. Balanced translocation of lOq andl3q, including the PTEN gene, in a boy with a human chorionic gonadotropin-secreting tumor and the Bannayan-Riley-Ruvalcaba syndrome.J Clin Endocrinol Metab.1999 Dec;84(12):4665-70
11. Wu LM, Li YL, Yin YH, Hou GQ, Zhu R, Hua XL, Xu JR, Chen ZA. Usefulness of dual-energy computed tomography imaging in the differential diagnosis of sellar meningiomas andpituitary adenomas:preliminary report.PLoS One.2014 Mar 3;9(3):e90658.
12.18. Cao X. R., Lill N. L., Boase N., Shi P. P., Croucher D. R., Shan H., Qu J., Sweezer E. M., Place T., Kirby P. A., Daly R. J., Kumar S., Yang B. (2008) Nedd4 controls animal growth by regulating IGF-1 signaling. Sci. Signal.1, ra5.
13.19. Treier M., Staszewski L. M., Bohmann D. (1994)Ubiquitin-dependent c-Jun degradation in vivo is mediated by the delta domain. Cell 78,787-798.
14.20. Chou T. C. (2006) Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol. Rev.58,621-681.
15.21. Franke T. F., Kaplan D. R., Cantley L. C., Toker A. (1997) Direct regulation of the Akt proto-oncogene product by phosphatidylinositol 3,4-bisphosphate. Science 275,665-668.
16. Wang L., Balas B., Christ-Roberts C. Y, Kim R. Y, Ramos F. J., Kikani C. K., Li C., Deng C., Reyna S., Musi N., Dong L. Q., DeFronzo R. A., Liu F. (2007) Peripheral disruption of the Grb 10 gene enhances insulin signaling and sensitivity in vivo. Mol. Cell Biol.27,6497-6505.
17.9. Wang X., Trotman L. C., Koppie T., Alimonti A., Chen Z., Gao Z., Wang J., Erdjument-Bromage H., Tempst P., Cordon-Cardo C., Pandolfi P. P., Jiang X. (2007)NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN. Cell 128, 129-139.
18. Yim E. K., Peng G, Dai H., Hu R., Li K., Lu Y., Mills G B., Meric-Bernstam F., Hennessy B. T., Craven R. J., Lin S. Y. (2009) Rak functions as a tumor suppressor by regulating PTEN protein stability and function. Cancer Cell15,304-314.
19. Drinjakovic J., Jung H., Campbell D. S., Strochlic L., Dwivedy A., Holt C. E. (2010) E3 ligase Nedd4 promotes axon branching by down-regulating PTEN. Neuron 65,341-357.
20. Trotman L. C., Wang X., Alimonti A., Chen Z., Teruya-Feldstein J., Yang H., Pavletich N. P., Carver B. S., Cordon-Cardo C., Erdjument-Bromage H., Tempst P., Chi S. G, Kim H. J., Misteli T., Jiang X., Pandolfi P. P. (2007)Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell 128,141-156.
21. Maccario H., Perera N. M., Gray A., Downes C. P., Leslie N. R. (2010)Ubiquitination of PTEN (phosphatase and tensin homolog) inhibits phosphatase activity and is enhanced by membrane targeting and hyperosmotic stress.J. Biol. Chem.285,12620-12628.
22. Wang X., Shi Y, Wang J., Huang G, Jiang X. (2008)Crucial role of the C terminus of PTEN in antagonizing NEDD4-1-mediated PTEN ubiquitination and degradation.Biochem. J.414,221-229.
23. Lee J. R., Oestreich A. J., Payne J. A., Gunawan M. S., Norgan A. P., Katzmann D. J. (2009) The HECT domain of the ubiquitin ligase Rsp5 contributes to substrate recognition. J. Biol. Chem.284,32126-32137.
24. Miller CR, Perry A.Glioblastoma. [J].Arch Pathol Lab Med.2007,131(3):397-406
25. Kim R H, Peters M, Jang Y, et al. DJ-1, a novel regulator of the tumor suppressor PTEN.[J]. Cancer Cell.2005,7(3):263-273.
26. Wu D N, Pei D S, Wang Q, et al. Down-regulation of PTEN by sodium orthovanadate inhibits ASK1 activation via PI3-K/Akt during cerebral ischemia in rat hippocampus.[J]. Neurosci Lett.2006,404(1-2):98-102.
27. Castellino RC, Durden DL.Mechanisms of Disease:the PI3K-Akt-PTEN signaling node--an intercept point for the control of angiogenesis in brain tumors.Nat Clin Pract Neurol.2007 Dec;3(12):682-93.
28. Mellinghoff IK, Cloughesy TF, Mischel PS.PTEN-mediated resistance to epidermal growth factor receptor kinase inhibitors.Clin Cancer Res.2007 Jan 15;13(2 Pt 1):378-81.
29. X, Trotman L C, Koppie T, et al. NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN.[J]. Cell.2007,128(1):129-139.
30. Rotman L C, Wang X, Alimonti A, et al. Ubiquitination regulates PTEN nuclear import and tumor suppression.[J]. Cell.2007,128(1):141-156.
1.张燕捷,房静远.介导抑癌蛋白泛素化的新分子,J MedMol Biol,2007,4 (5):431-433
2. Wang X, Trotman L C, Koppie T, et al. NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN. Cell.2007,128(1):129-139.
3. Ciechanover A. The ubiquitin proteolytic system. Neurology,2006,66 (Supp 1 1):17-19.
4. Fire A, Xu S, Montgomery MK, et al. Potent and sepecific genetic interference by doule-stranded RNA in Caenorhabditis elegans[J]. Nature,1998,391: 8063-8067.
5. atzkem,Matzke.A,J.Kooter,J.M. RNA:guiding gene silencing [J] Science, 2001,293 (5532):1080-1085.
6. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells[J]. Science,2002, 296(5567):550-553.
7. Mottiml,Califanod,Baldassarreg, etal. Reduced E-cadherin expression contributes to the loss of p27kipl mediated apparatus of contact inhibition in thyroid anap lastic carcinomas. Carcinogenesis,2005,26 (6):1021-1034.
8. Gagliano T, Filieri C, Minoia M, Buratto M, Tagliati F, et al. (2013) Cabergoline reduces cell viability in non functioning pituitary adenomas by inhibiting vascular endothelial growth factor secretion. Pituitary 16:91-100.
9. Donangelo I, Gutman S, Horvath E, Kovacs K, Wawrowsky K, et al. (2006) Pituitary tumor transforming gene overexpression facilitates pituitary tumor development.Endocrinology 147:4781-4791.
10. Gruppetta M, Mercieca C, Vassallo J (2013) Prevalence and incidence of pituitary adenomas:a population based study in Malta. Pituitary 16:545-553.
11. Kleihues P, Louis DN, Scheithauer BW, Rorke LB, Reifenberger G et al. (2002) The WHO classification of tumors of the nervous system. J Neuropathol Exp Neurol 61:215-226
12. Simonin A, Fuster D (2010)Nedd4-1 and beta-arrestin-1 are key regulators of Na+/H+exchanger 1 ubiquitylation, endocytosis, and function. J Biol Chem 285:38293-38303.
13. Zhang H, Nie W, Zhang X, Zhang G, Li Z, Wu H, Shi Q, Chen Y, Ding Z, Zhou X, Yu R. NEDD4-1 regulates migration and invasion of glioma cells through CNrasGEF ubiquitination in vitro.PLoS One.2013 Dec 10;8(12):e82789.
14. Jung S, Li C, Jeong D, Lee S, Ohk J, Park M, Han S, Duan J, Kim C, Yang Y, Kim KI, Lim JS, Kang YS, Lee MS. Oncogenic function of p34SEI-1 via NEDD4-1-mediated PTEN ubiquitination/degradation and activation of the PI3K/AKT pathway. Int J Oncol.2013 Nov;43(5):1587-95.
15. Bobby R, Medini K, Neudecker P, Lee TV, Brimble MA, McDonald FJ, Lott JS, Dingley AJ. Structure and dynamics of human Nedd4-1 WW3 in complex with the aENaC PY motif.Biochim Biophys Acta.2013 Aug;1834(8):1632-41
16. Calalb MB, Polte TR, Hanks SK. Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity:a role for Src family kinases [J]. Mol Cell Biol,1995,15 (2):954-963.
17. DJ Sieg, CR Hauck, DD Schlaepfer. Required role of focal adhesion kinase (FAK) for integrin-stimulated cellmigration [J].J Cell Sci,1999,112 (16) 2677-2691.
18. Noguchi K, Yamana H, Kitanaka C.et al. Differential role of the JNKand p38 MAPKpathway in c-Myc and s-Myc mediated apoptosis [J]. Biochem Biophys ResCommun,2000; 267 (1):221-227.
19. Han L, Wang P, Zhao G, Wang H, Wang M, Chen J, Tong T. Upregulation of SIRT1 by 17β-estradiol depends on ubiquitin-proteasome degradation of PPAR-γ mediated byNEDD4-l.Protein Cell.2013 Apr;4(4):310-21.
20. Fan CD, Lum MA, Xu C, Black JD, Wang X. Ubiquitin-dependent regulation of phospho-AKT dynamics by the ubiquitin E3 ligase, NEDD4-1, in the insulin-like growth factor-1 response.J Biol Chem.2013 Jan 18;288(3):1674-84
21. Howitt J., Lackovic J., Low L. H., Naguib A., Macintyre A., Goh C. P., Callaway J. K., Hammond V., Thomas T., Dixon M., Putz U., Silke J., Bartlett P., Yang B., Kumar S., Trotman L. C., Tan S. S. (2012)Ndfip1 regulates nuclear PTEN import in vivo to promote neuronal survival following cerebral ischemia. J. Cell Biol.196,29-36.
22. Fouladkou F., Landry T., Kawabe H., Neeb A., Lu C., Brose N., Stambolic V., Rotin D. (2008) The ubiquitin ligase Nedd4-1 is dispensable for the regulation of PTEN stability and localization. Proc. Natl. Acad. Sci. U.S.A.105, 8585-8590.
23.18. Cao X. R., Lill N. L., Boase N., Shi P. P., Croucher D. R., Shan H., Qu J., Sweezer E. M., Place T., Kirby P. A., Daly R. J., Kumar S., Yang B. (2008) Nedd4 controls animal growth by regulating IGF-1 signaling. Sci. Signal. 1,ra5.
24. modification of the 20S proteasomal proteins of the archaeon Haloferax volcanii. J Bacteriol,2006,188(21):7521
25. Han L, Wu C, Riaz H, Bai L, Chen J, Zhen Y, Guo A, Yang L. Characterization of the mechanism of inhibin a-subunit gene in mouse anterior pituitary cells by RNA interference.PLoS One.2013 Oct 3;8(10)
26. Huang SQ, Liao QJ, Wang XW, Xin DQ, Chen SX, Wu QJ, Ye G. RNAi-mediated knockdown of pituitary tumor-transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells.Braz J Med Biol Res.2012 Nov;45(11):995-1001.
27. Vassalli JD, Pepper MS. Tumour biology. Membrane proteases in focus. [J]. Nature,1994,370(6484):14-5.
28. Cockett MI, Birch ML, Murphy q Hart IR, Docherty AJ. Metalloproteinase domain structure, cellular invasion and metastasis [J].Biochem Soc Trans,1994, 22(1):SS-7.
29. Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, Seiki M. A matrix metal oproteinase expressed on the surface of invasive tumor cells [J].Narure,1994,370:61-65.
2. Palumbo T, Faucz FR, Azevedo M, Xekouki P, Iliopoulos D, Stratakis CA: Functional screen analysis reveals miR-26b and miR-128 as central regulators of pituitary somatomammotrophic tumor growth through activation of the PTEN-AKT pathway.Oncogene.2013 Mar 28;32(13):1651-9
3. Wang X, Trotman L C, Koppie T, et al. NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN.[J]. Cell.2007,128(1):129-139.
4. Wu D N, Pei D S, Wang Q, et al. Down-regulation of PTEN by sodium orthovanadate inhibits ASK1 activation via PI3-K/AKT during cerebral ischemia in rat hippocampus.[J]. Neurosci Lett.2006,404(1-2):98-102.
5. Castellino RC, Durden DL.Mechanisms of Disease:the PI3K-AKT-PTEN signaling node--an intercept point for the control of angiogenesis in brain tumors.Nat Clin Pract Neurol.2007 Dec;3(12):682-93.
6. Tena-Suck ML, Ortiz-Plata A, de la Vega HA. Phosphatase and tensin homologue and pituitary tumor-transforming gene in pituitary adenomas. Clinical-pathologic and immunohistochemical analysis.Ann Diagn Pathol.2008 Aug;12(4):275-82.
7. Feng ZZ, Chen JW, Yang ZR, Lu GZ, Cai ZG Expression of PTTG1 and PTEN in endometrial carcinoma:correlation with tumorigenesis and progression.Med Oncol. 2012 Mar;29(1):304-10
8. Vivanco I & Sawyers CL 2002 The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nature Review Cancer 2 489-501.
9. Whiteman DC, Zhou XP, Cummings MC, Pavey S, Hayward NK & Eng C 2002Nuclear PTEN expression and clinicopathologic features in a population-based series of primary cutaneous melanoma. International Journal of Cancer 9963-67.
10. Faisal Ahmed S, Marsh DJ, Weremowicz S, Morton CC, Williams DM, Eng C. Balanced translocation of lOq andl3q, including the PTEN gene, in a boy with a human chorionic gonadotropin-secreting tumor and the Bannayan-Riley-Ruvalcaba syndrome.J Clin Endocrinol Metab.1999 Dec;84(12):4665-70
11. Wu LM, Li YL, Yin YH, Hou GQ, Zhu R, Hua XL, Xu JR, Chen ZA. Usefulness of dual-energy computed tomography imaging in the differential diagnosis of sellar meningiomas andpituitary adenomas:preliminary report.PLoS One.2014 Mar 3;9(3):e90658.
12.18. Cao X. R., Lill N. L., Boase N., Shi P. P., Croucher D. R., Shan H., Qu J., Sweezer E. M., Place T., Kirby P. A., Daly R. J., Kumar S., Yang B. (2008) Nedd4 controls animal growth by regulating IGF-1 signaling. Sci. Signal.1, ra5.
13.19. Treier M., Staszewski L. M., Bohmann D. (1994)Ubiquitin-dependent c-Jun degradation in vivo is mediated by the delta domain. Cell 78,787-798.
14.20. Chou T. C. (2006) Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol. Rev.58,621-681.
15.21. Franke T. F., Kaplan D. R., Cantley L. C., Toker A. (1997) Direct regulation of the Akt proto-oncogene product by phosphatidylinositol 3,4-bisphosphate. Science 275,665-668.
16. Wang L., Balas B., Christ-Roberts C. Y, Kim R. Y, Ramos F. J., Kikani C. K., Li C., Deng C., Reyna S., Musi N., Dong L. Q., DeFronzo R. A., Liu F. (2007) Peripheral disruption of the Grb 10 gene enhances insulin signaling and sensitivity in vivo. Mol. Cell Biol.27,6497-6505.
17.9. Wang X., Trotman L. C., Koppie T., Alimonti A., Chen Z., Gao Z., Wang J., Erdjument-Bromage H., Tempst P., Cordon-Cardo C., Pandolfi P. P., Jiang X. (2007)NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN. Cell 128, 129-139.
18. Yim E. K., Peng G, Dai H., Hu R., Li K., Lu Y., Mills G B., Meric-Bernstam F., Hennessy B. T., Craven R. J., Lin S. Y. (2009) Rak functions as a tumor suppressor by regulating PTEN protein stability and function. Cancer Cell15,304-314.
19. Drinjakovic J., Jung H., Campbell D. S., Strochlic L., Dwivedy A., Holt C. E. (2010) E3 ligase Nedd4 promotes axon branching by down-regulating PTEN. Neuron 65,341-357.
20. Trotman L. C., Wang X., Alimonti A., Chen Z., Teruya-Feldstein J., Yang H., Pavletich N. P., Carver B. S., Cordon-Cardo C., Erdjument-Bromage H., Tempst P., Chi S. G, Kim H. J., Misteli T., Jiang X., Pandolfi P. P. (2007)Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell 128,141-156.
21. Maccario H., Perera N. M., Gray A., Downes C. P., Leslie N. R. (2010)Ubiquitination of PTEN (phosphatase and tensin homolog) inhibits phosphatase activity and is enhanced by membrane targeting and hyperosmotic stress.J. Biol. Chem.285,12620-12628.
22. Wang X., Shi Y, Wang J., Huang G, Jiang X. (2008)Crucial role of the C terminus of PTEN in antagonizing NEDD4-1-mediated PTEN ubiquitination and degradation.Biochem. J.414,221-229.
23. Lee J. R., Oestreich A. J., Payne J. A., Gunawan M. S., Norgan A. P., Katzmann D. J. (2009) The HECT domain of the ubiquitin ligase Rsp5 contributes to substrate recognition. J. Biol. Chem.284,32126-32137.
24. Miller CR, Perry A.Glioblastoma. [J].Arch Pathol Lab Med.2007,131(3):397-406
25. Kim R H, Peters M, Jang Y, et al. DJ-1, a novel regulator of the tumor suppressor PTEN.[J]. Cancer Cell.2005,7(3):263-273.
26. Wu D N, Pei D S, Wang Q, et al. Down-regulation of PTEN by sodium orthovanadate inhibits ASK1 activation via PI3-K/Akt during cerebral ischemia in rat hippocampus.[J]. Neurosci Lett.2006,404(1-2):98-102.
27. Castellino RC, Durden DL.Mechanisms of Disease:the PI3K-Akt-PTEN signaling node--an intercept point for the control of angiogenesis in brain tumors.Nat Clin Pract Neurol.2007 Dec;3(12):682-93.
28. Mellinghoff IK, Cloughesy TF, Mischel PS.PTEN-mediated resistance to epidermal growth factor receptor kinase inhibitors.Clin Cancer Res.2007 Jan 15;13(2 Pt 1):378-81.
29. X, Trotman L C, Koppie T, et al. NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN.[J]. Cell.2007,128(1):129-139.
30. Rotman L C, Wang X, Alimonti A, et al. Ubiquitination regulates PTEN nuclear import and tumor suppression.[J]. Cell.2007,128(1):141-156.
1.张燕捷,房静远.介导抑癌蛋白泛素化的新分子,J MedMol Biol,2007,4 (5):431-433
2. Wang X, Trotman L C, Koppie T, et al. NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN. Cell.2007,128(1):129-139.
3. Ciechanover A. The ubiquitin proteolytic system. Neurology,2006,66 (Supp 1 1):17-19.
4. Fire A, Xu S, Montgomery MK, et al. Potent and sepecific genetic interference by doule-stranded RNA in Caenorhabditis elegans[J]. Nature,1998,391: 8063-8067.
5. atzkem,Matzke.A,J.Kooter,J.M. RNA:guiding gene silencing [J] Science, 2001,293 (5532):1080-1085.
6. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells[J]. Science,2002, 296(5567):550-553.
7. Mottiml,Califanod,Baldassarreg, etal. Reduced E-cadherin expression contributes to the loss of p27kipl mediated apparatus of contact inhibition in thyroid anap lastic carcinomas. Carcinogenesis,2005,26 (6):1021-1034.
8. Gagliano T, Filieri C, Minoia M, Buratto M, Tagliati F, et al. (2013) Cabergoline reduces cell viability in non functioning pituitary adenomas by inhibiting vascular endothelial growth factor secretion. Pituitary 16:91-100.
9. Donangelo I, Gutman S, Horvath E, Kovacs K, Wawrowsky K, et al. (2006) Pituitary tumor transforming gene overexpression facilitates pituitary tumor development.Endocrinology 147:4781-4791.
10. Gruppetta M, Mercieca C, Vassallo J (2013) Prevalence and incidence of pituitary adenomas:a population based study in Malta. Pituitary 16:545-553.
11. Kleihues P, Louis DN, Scheithauer BW, Rorke LB, Reifenberger G et al. (2002) The WHO classification of tumors of the nervous system. J Neuropathol Exp Neurol 61:215-226
12. Simonin A, Fuster D (2010)Nedd4-1 and beta-arrestin-1 are key regulators of Na+/H+exchanger 1 ubiquitylation, endocytosis, and function. J Biol Chem 285:38293-38303.
13. Zhang H, Nie W, Zhang X, Zhang G, Li Z, Wu H, Shi Q, Chen Y, Ding Z, Zhou X, Yu R. NEDD4-1 regulates migration and invasion of glioma cells through CNrasGEF ubiquitination in vitro.PLoS One.2013 Dec 10;8(12):e82789.
14. Jung S, Li C, Jeong D, Lee S, Ohk J, Park M, Han S, Duan J, Kim C, Yang Y, Kim KI, Lim JS, Kang YS, Lee MS. Oncogenic function of p34SEI-1 via NEDD4-1-mediated PTEN ubiquitination/degradation and activation of the PI3K/AKT pathway. Int J Oncol.2013 Nov;43(5):1587-95.
15. Bobby R, Medini K, Neudecker P, Lee TV, Brimble MA, McDonald FJ, Lott JS, Dingley AJ. Structure and dynamics of human Nedd4-1 WW3 in complex with the aENaC PY motif.Biochim Biophys Acta.2013 Aug;1834(8):1632-41
16. Calalb MB, Polte TR, Hanks SK. Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity:a role for Src family kinases [J]. Mol Cell Biol,1995,15 (2):954-963.
17. DJ Sieg, CR Hauck, DD Schlaepfer. Required role of focal adhesion kinase (FAK) for integrin-stimulated cellmigration [J].J Cell Sci,1999,112 (16) 2677-2691.
18. Noguchi K, Yamana H, Kitanaka C.et al. Differential role of the JNKand p38 MAPKpathway in c-Myc and s-Myc mediated apoptosis [J]. Biochem Biophys ResCommun,2000; 267 (1):221-227.
19. Han L, Wang P, Zhao G, Wang H, Wang M, Chen J, Tong T. Upregulation of SIRT1 by 17β-estradiol depends on ubiquitin-proteasome degradation of PPAR-γ mediated byNEDD4-l.Protein Cell.2013 Apr;4(4):310-21.
20. Fan CD, Lum MA, Xu C, Black JD, Wang X. Ubiquitin-dependent regulation of phospho-AKT dynamics by the ubiquitin E3 ligase, NEDD4-1, in the insulin-like growth factor-1 response.J Biol Chem.2013 Jan 18;288(3):1674-84
21. Howitt J., Lackovic J., Low L. H., Naguib A., Macintyre A., Goh C. P., Callaway J. K., Hammond V., Thomas T., Dixon M., Putz U., Silke J., Bartlett P., Yang B., Kumar S., Trotman L. C., Tan S. S. (2012)Ndfip1 regulates nuclear PTEN import in vivo to promote neuronal survival following cerebral ischemia. J. Cell Biol.196,29-36.
22. Fouladkou F., Landry T., Kawabe H., Neeb A., Lu C., Brose N., Stambolic V., Rotin D. (2008) The ubiquitin ligase Nedd4-1 is dispensable for the regulation of PTEN stability and localization. Proc. Natl. Acad. Sci. U.S.A.105, 8585-8590.
23.18. Cao X. R., Lill N. L., Boase N., Shi P. P., Croucher D. R., Shan H., Qu J., Sweezer E. M., Place T., Kirby P. A., Daly R. J., Kumar S., Yang B. (2008) Nedd4 controls animal growth by regulating IGF-1 signaling. Sci. Signal. 1,ra5.
24. modification of the 20S proteasomal proteins of the archaeon Haloferax volcanii. J Bacteriol,2006,188(21):7521
25. Han L, Wu C, Riaz H, Bai L, Chen J, Zhen Y, Guo A, Yang L. Characterization of the mechanism of inhibin a-subunit gene in mouse anterior pituitary cells by RNA interference.PLoS One.2013 Oct 3;8(10)
26. Huang SQ, Liao QJ, Wang XW, Xin DQ, Chen SX, Wu QJ, Ye G. RNAi-mediated knockdown of pituitary tumor-transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells.Braz J Med Biol Res.2012 Nov;45(11):995-1001.
27. Vassalli JD, Pepper MS. Tumour biology. Membrane proteases in focus. [J]. Nature,1994,370(6484):14-5.
28. Cockett MI, Birch ML, Murphy q Hart IR, Docherty AJ. Metalloproteinase domain structure, cellular invasion and metastasis [J].Biochem Soc Trans,1994, 22(1):SS-7.
29. Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, Seiki M. A matrix metal oproteinase expressed on the surface of invasive tumor cells [J].Narure,1994,370:61-65.