The Study Of The Expression And Effection Of NEDD4-1/PTEN In Pituitary Adenomas

Part one:The study of the expression and correlation of NEDD4-1and PTEN in pituitary adenomasBackgroundPituitary 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 cellsObjective 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.