| Thesis I The expression of SPARC in human intracranial aneurysms and its relationship with MMP-2/-9BACKGROUND:Intracranial aneurysm (IA) is a tumor-like protrusions on the intracranial arterial wall, which is also the leading cause of spontaneous subarachnoid hemorrhage, and aneurysm rupture have a high fatality rate and disability. SPARC (Secreted Protien, Acidic and Rich in Cysteine), which is rich in cysteine (Cys) anti-adhesion acid glycoprotein, also known as osteonectin or BM-40. It can express a variety of tissue cells including endothelial cells, vascular smooth muscle cells (vascular smooth nuscles cells, VSMCs) as well as fibroblasts, also plays an important role in tissue remodeling, cell proliferation, as well as the body of the pathophysiologic process, increasing attention has been paid by many scholars. The development of intracranial aneurysms need the original blood vessels to dilate, increased permeability of endothelial cell proliferation, migration, extracellular matrix degradation, and other pathological changes, however, the degradation of the extracellular matrix is a key step in the development of intracranial aneurysms. Matrix metalloproteinase enzymes (MMPs) are the most important protease involved in extracellular matrix degradation, and its ability to degrade all known extracellular matrix components, also we all know that MMP-2/-9 is the most important matrix metalloproteinases, and closely related to the aneurysm. To blood flow stress injury, there is a dynamic process that is damage/repair in intracranial arteries, and SPARC can influence this balance, which means that the formation of intracranial aneurysms is mainly because of the broken of this dynamic balance, therefore, SPARC may play an important role in the occurrence of intracranial aneurysms. Explore the relationship between the aneurysms and SPARC MMP-2/-9 protein expression has important clinical significance in the study of formation mechanism of aneurysm. In this paper, we first summarized the clinical manifestations and pathological features of patients with intracranial aneurysms, then detected the expression and distribution of SPARC and MMP-2 and MMP-9 in specimens of patients with intracranial aneurysms by immunohistochemistry, and explore the role of SPARC in the pathogenesis of intracranial aneurysms by study the function of SPARC and MMP-2 and MMP-9 protein, and expect the SPARC to be meaningful biological markers to assess the formation and rupture of intracranial aneurysms.OBJECTIVE:The goal of this study was to establish the role of SPARC in human intracranial aneurysms.METHOD:Thirty-one intracranial aneurysms were immunohistochemically and HE stained for SPARC, MMP-2 and MMP-9. As controls, normal Circle of Willis arteries were similarly immunostained. All specimens were retrieved during autopsies and were embedded in paraffin. To evaluate the expression levels of SPARC, MMP-2 and MMP-9, western blotting was also performed in three available intracranial aneurysm specimens. The limited availability of fresh intracranial aneurysm tissue was the result of the majority of patients choosing endovascular embolization.RESULTS:1 Results of histological enzyme stainingHE staining showed that the light microscope, aneurysm (full thickness) could barely see the smooth, elastic fibers absent, replaced by collagen fibers; sometimes see the parietal tissue necrosis and inflammatory cell infiltration, intraluminal thrombus visible; aneurysm sometimes there is a small amount of bleeding; often mural thrombus, intimal thickening fibers (collagen fibers); medial atrophy, elastic fiber breakage/fibrosis/calcification, fibrous outer membrane hyperplasia; chronic inflammation2 Immunohistochemical staining showed that the expression of SPARC, mmp-2 and mmp-9 in the aneurysm tissue is highIt was found that normal the expression of SPARC, mmp-2 and mmp-9intracranial willis arterial showed low or no positive staining; but SPARC, mmp-2 and mmp-9 showed strong staining in intracranial aneurysm. Also observed, regardless of unruptured aneurysms and ruptured aneurysm, SPARC, mmp-2 and mmp-9 showed moderate to high intensity of staining. Statistical analysis showed that there is statistically significant between SPARC staining and mmp-2 and mmp-9(p<0.001).3 Western-blot analysis showed that the expression of SPARC, mmp-2 and mmp-9 in higher in the intracranial aneurysm than normal arteryIn order to analyze expression levels of SPARC, mmp-2 and mmp-9 in intracranial aneurysms, we chose three fresh cases of intracranial aneurysms (currently very difficult to get surgery) specimens. The results showed that, compared with normal willis arterial, the expression of SPARC, mmp-2 and mmp-9 significantly increased in aneurysms.CONCLUSION:The expression of SPARC, MMP-2/-9 protein is closely related with the occurrence of intracranial aneurysms, and SPARC protein may influence formation and progress of aneurysm through the regulation of expression of extracellular matrix proteins; the rupture of extracellular matrix may be the pathophysiological process of the formation of intracranial aneurysms.SIGNFICANCE:By studying this subject, specifically in intracranial aneurysm SPARC expression and correlation, and clearly it can regulate MMP-2/-9 expression, providing a new potential target for intracranial aneurysms are closely related.Thesis II analyze the correlation between the expression of FAP-1 and glioblastoma based on ONCOMINE database and experimental studies on its role in the cell viability, invasion and migrationBACKGROUND:Glioblastoma Multiforme (GBM) is a highly aggressive primary brain tumour with almost complete patient mortality attributing to its invasive nature and destruction of surrounding brain tissue. Despite progression of numerous drug clinical trials for GBM treatment, few objective responses are observed and the median survival of 15 months has increased little over the past decade. Our understanding of key molecular mechanisms to GBM tumour progression, however, has exploded during this time but we still fail to make significant clinical progress. By now, most studies involving molecular neurobiology gliomas are focusing on ways to growth factor receptor tyrosine kinase (PTK) and phosphatidylinositol phosphatase signaling pathways. And a tyrosine kinase, protein tyrosine phosphatase phosphorylation state regulation of many important signaling molecules, although compared to the tyrosine kinase for their research little is known. PTP family as another important protein is FAP-1 (PTPN13/PTPL1), also known as FAS-related phosphorylase (FAS-associated phosphatase 1) is closely associated with tumor development.In addition, there is evidence that, FAP-1 may be a tumor suppressor gene. For example, in the stomach and liver cancer has been observed that by promoter hypermethylation or allelic loss can reduce the PTPN13 mRNA expression, and FAP-1 knockdown improve PLC5 cell proliferation. In a large-scale mutation analysis of colorectal cancer in a PTP display, found 19 PTPN13 mutations,7 in PTP domain, which may impair the catalytic activity of FAP-1 in 29. Meanwhile, FAP-1 is one of the largest intracellular PTPs, which indicates that it has a variety of functions and may be in the presence of a context-dependent play a positive or negative mode of action. Therefore, FAP-1 resistance to cancers is a promising therapeutic target. The main purpose of this experiment is to clear the role of FAP-1 in GBM progression and chemotherapy resistance.OBJECTIVE:1 To analysis oncomine database, understand the role of FAP-1 to the correlation with GBM.2 Observe the role of FAP-1 in GBM cells viability, invasion and migration and related molecular mechanism.3 Test the role of FAP-1 in GBM cells chemotherapy resistance.METHOD:1 UCSC genome database to detect the expression of FAP-1 mRNAWe analyzed 16 phosphatases in glioma and normal brain tissue expression through oncomine, which is set by each data to the threshold criteria for inclusion in the analysis to achieve. Initially the threshold criteria for this study is a p-value of<0.05 and mRNA expression fold change> 1.4. Fold change is classified changes in cancer tissues and normal tissues and thus detect mRNA expression levels of the target gene. Based on the threshold criteria, Oncomine all genes in all data sets will get a gene ranking. Image data is obtained with respect to the target gene based on the gene of interest than% p-value p-value for all other genes within the same data set on. Gene expression data obtained from the oncomine is based on the number and the standard deviation normalized to convert each array studies to achieve.2 The effect of FAP-1 on GBM cell viabilityPreparation of different concentrations (0,6.25nm,12.5nm,25nm) cell suspension, 96-well plates were inoculated cells/about 1000 per well, placing 37℃, cultured CO2, the first two days to replace the culture medium, returned to the incubator cultivation on 7 days, the micropipette add 40ul CV buffer/per well into 96-well plates at room temperature for 10 minutes, the micropipette taking 30ul/per well into the Celltiter-Glo96 orifice same position, GloMax 96 micro orifice luminescence detector test.3 The effect of FAP-1 on GBM cell migrationCell scratch test the role of FAP-1 on cell migration after siRNA, using more accurate cell microelectronic chip detection technology (xCELLLigence) to detecte the influence of FAP-1 on cell migration.4 The effect of FAP-1 on mmp-2 activityWestern-blot analysis expression of mmp-2 after FAP-1 transfection; gelatin zymography test the activity of mmp-2 in cell culture supernatant.5 The effect of FAP-1 on GBM chemotherapy resistanceUse (Onm,6.25nm,12.5nm,25nm) different concentrations of siRNA transfected cells, after use (OGy,1.25 Gy,2.5 Gy) with different doses of radiation and different doses (Oum,125 um,250 um,500 um,1000 um) of temozolomide treated cells, cell viability test cell activity, the data recording and statistical analysis.6 The effect of FAP-1 on other signaling pathways(AKT/ERK1/2)Use different concentrations (Oul, 1ul,4ul) of siRNA transfected cells, western-blot detection the expression of p-AKT, t-AKT, p-ERK1/2, t-ERK1/2. RESULTS:1 Oncomine database analysis FAP-1 is closely related to the GBMWe evaluated the mRNA expression of PTPN13/FAP-1 across a number of cancer types comparing tumour versus normal tissue.6 out of 9 studies evaluated showed enhanced PTPN13/FAP-1 mRNA expression in GBM vs normal brain, while 3 studies showed no difference. This data suggest overall that PTPN13/FAP-1 expression may correlates with tumour development and progression but is not conclusive.2 Knockdown of FAP-1 can enhanced GBM cell activityTransfected with increasing concentration found LN18 and U87MG cell activity groups were gradually increased, however, by multiple sets of ANOVA analysis found that only the concentration of 6.25nm statistically significant (p＜0.001). These results suggest that, FAP-1-mediated cell activity can also reflect its function in a dose-dependent manner.3 Knockdown of FAP-1 can enhanced cell migration GBMCell scratch results found that as the concentration of transfection enhancement, U87MG group significantly increased cell migration, but LN18 group only 25nm at which enhanced cell migration, cell migration remaining concentration but decreased, which may be related to experimental variation. Cell microelectronic chip detection technology (xCELLLigence) experimental results confirmed LN18 group compared with the control group, the transfected group mobility enhancement 33%(P<0.001); U87MG group compared with the control group, transfection group migration rates are also enhanced 42%(P<0.001).4 Knockdown of FAP-1 can enhanced the activity of mmp-2 in GBM cellsGelatinase spectrum results show that with the increase of the concentration of transfection, mmp-2 activity LN18 group and U87MG group gradually increased, but with different concentrations of transfected mmp-2 activity increased in both groups in varying degrees.5 FAP-1 reduction led to increased resistance to radiotherapy and temozolomideCell viability was found that in In LN18 group, radiation group, temozolomide group and transfection group was statistically significant (p=0.002), radiation group and temozolomide group showed no statistically significant; in U87MG group, temozolomide group and transfection group and radiation group were statistically significant (p<0.001), radiation group and transfection group, temozolomide group and transfection group were significantly related effects (p<0.001) western-blot results showed that, LN18 and U87MG cells were appeared as for the increase in temozolomide concentration, the expression of FAP-1 is gradually decreased, while, FAP-1 in day 3 was significantly lower than in the first day of expression, suggesting that FAP-1 can be temozolomide-induced inactivation.6 Western-blot analysis of AKT and ERK1/2 phosphorylation status after knockdown of FAP-1It was found that two groups of cells, p-AKT, the expression of p-ERK1/2 is decreased, and after EGF treated cells, the expression of p-AKT, p-ERK1/2 is also enhanced to control state. These results indicated that a certain degree, FAP-1 can affect the phosphorylation status by AKT and ERKl/2 to modulate biological activity of cells, while described, there may be additional signaling proteins involved in achieving the function of FAP-1, but the specific mechanism needs further study confirmedCONCLUSION:1 FAP-1 mRNA is high expression in glioblastoma, and is good related with it.2 FAP-1 inactivation can enhance the activity of glioblastoma cell lines, enhance cell migration, enhanced cell invasion.3 FAP-1 inactivation can enhance GBM cells resistant to treatment, suggesting FAP-1 inactivation may be one of the important factor on GBM poor efficacy and adverse prognostic.SIGNIFANCE:This experiment oncomine database analysis on a wide range of protein phosphatase associated with glioblastoma is the first time the international community, which provides for the future more people involved in the relationship between the gene and tumor progression of big data analysis purposes an important basis; FAP-1 may be a closely related malignant glioblastoma biology degree marker protein... |
