| Gliomas are the most common primary brain tumor and accounts for approximately50%of all brain tumors, and originated mostly from astrocytes, oligodentrocytes andependymal cells. As we all known, nearly70%are malignant gliomas, and most of themare originated from astrocytes, such as glioblastoma and anaplastic astrocytoma, and ofpoor prognosis. It is reported that the formation and the recurrence of glioma areinfluenced by different signaling pathways which contained several genes, proteins andcytokines. The molecular mechanisms underlying the initiation, maintenance andprogression of glioblastoma still remain largely unclarified. Recent understanding ingenomic and molecular abnormalities in glioma has shifted the treatment paradigmtowards using molecularly targeted agents and allowed more rational uses of targetedmolecular therapies.Golgi phosphoprotein3(GOLPH3), is encoded by a gene residing on humanchromosome5p13, and located in the trans-Golgi network. GOLPH3is implicated inprotein trafficking, receptor recycling and glycosylation from Golgi to plasma membrane.Recent studies have shown GOLPH3could promote cellular transformation and tumorgrowth. Specifically, GOLPH3is highly expressed in several solid tumors, includingmelanoma, colon adenocarcinoma and non-small-cell lung cancer. GOLPH3enhancesmammalian target of rapamycin (mTOR) signaling in human cancer cells, and modulatescellular response to mTOR inhibitors. mTOR, a serine/threonine protein kinase and ‘targetof rapamycin’, acts as a primary regulator of protein synthesis and cell growth, and functions as a key integrator of the receptor tyrosine kinases and phosphatidyl-inositol-3kinases (RTK-PI3K) pathway, which in turn is the most frequently hyperactivated pathwayin glioma. Recent study has shown that in mRNA and protein level, GOLPH3expressionin human glioma is associated with the pathological grade, and no expression in normalbrain. However, the relationship between GOLPH3and the development and proliferationand invasion of glioma was still unknown. The aim of this study is to clarify the relationshipbetween the expression of GOLPH3glioma tissues and the outcome of glioma patients bytissue micorarray; and also clarify the exact role for GOPH3in the oncogenic process ofglioma cells by knocking down or overexpression its expression level in U251and U87celllines.Bone sialoprotein (BSP), a specific regulator of matrix metalloproteinase-2(MMP-2),is highly expressed in numerous cancers such as prostate cancer, breast cancer, andpancreatic cancer and may contribute to their invasive potential. BSP may also act as apro-angiogenic factor to promote angiogenesis through binding to integrin αvβ3. However,there has been no study on the role in the the development and proliferation and invasionof glioma and the expression of BSP in gliomas. In the current study, we sought toexamine the expression of BSP in human glioma tissue and normal brain tissues byimmunohistochemistry using tissue microarray and find out the correlation between BSPexpression and prognosis of glioma patients.Part I: Construct the glioma tissue microarrayPurpose: By using of paraffin wax of glioma specimens to construct the tissue microarray (TMA), and the method of immunohistochemistry, we studied special targets inthe glioma tissues to find out the relationship between the expressions of the target and thepathological grade and the prognosis of glioma patients.Method:1）All300glioma tissue specimens were obtained from glioma patients whounderwent surgical treatment at the Department of Neurosurgery, Changzheng Hospitalbetween January2000and November2010. The16normal brain tissues were obtainedfrom the patients who need the decompression surgery;2）The follow-up was carried out inall patients, and the survival time was censored in September2011.4）Cumulative survivalwas calculated by the Kaplan-Meier method and analyzed by log-rank test. Univariate andmultivariate analyses were performed by stepwise forward Cox regression model (P<0.2was considered as the inclusion criterion for factors that could be added into multivariateanalysis). All statistical analyses were performed with the SPSS16.0software (SPSS Inc,Chicago, IL), P<0.05was considered as statistically significantly different.Results: The tissue microarray contains: WHO grade I:9pilocytic astrocytomas,1Subependymal giant cell astrocytoma,1Subependymoma; WHO grade II:67Astrocytomas,27oligodendrocytomas,7oligoastrocytomas,7ependymomas,1papillomachoroideum; WHO grade III:33anaplastic astrocytomas,11anaplasticoligodendrocytomas,2anaplastic ependymomas,1astroblastoma; WHO grade IV:119glioblastomas,12medulloblastomas,1gliosarcoma,1PNET. By follow-up, we found thatin all300glioma patients,66.3%patients were male;85%patients showed younger agethan60years; the elipsy were observed in80.3%patients; necrosis on MRI was observed in12.7%patients.78.7%patients underwent total resection.62%patients receivedadjuvant chemotherapy and64.7%patients underwent adjuvant radiotherapy. We obtained284results of follow-up: the median overall survival was30months (95%CI：21.8-38.2months); The median progression-free survival was25months (95%CI:19.5-30.5months).The5years OS was34%, and the5years PFS is28%.Conclusion: Tissue microarray is an analysis tool for large sample of tumor. We canobtained the result of prognosis of patients by retrospectively analyzes combining withstatistical tools. Part Ⅱ: Study the relationship between the GOLPH3’sexpression and the prognosis of glioma patients and the role ofGOLPH3in glioma cells in vitroPurpose: GOLPH3, as an important protein in mTOR signaling, is overexpressed inand correlates with the pathological grade of glioma. In the current study, we examined theexpression of GOLPH3in gliomas with tissue microarray and correlated the measure to thepatient outcome.Method:1) GOLPH3expression in tumor tissue from300glioma patients wasexamined with tissue microarray and immunohistochemistry. Potential effects of GOLPH3on tumor growth were also examined in representative cell lines (U251and U87) by downregulating GOLPH3with RNA interference.Results: In all300glioma samples, we obtained284results of follow-up. In284glioma samples, we adopted270glioma samples for analyzing (the other14tissue dotswere lost from the TMA slide). In270glioma patients, the univariate analysis showed nosignificance between the expression of GOLPH3and the prognosis of glioma patients(p=0.698，p=0.704). In all106GBM samples, we adopted the97promary GBM samplesfor analyzing (the other9tissue dots were lost from the TMA slide). Tissue microarrayanalysis revealed high GOLPH3expression in40patients (40/97,41.2%) and lowGOLPH3expression in the remaining57patients (57/97,58.8%).We obtained93GBMpatients’ follow-up. In93GBM patients, the median overall survival (OS) was12months(95%CI:10.31-13.69months). The median progression-free survival (PFS) was10months(95%CI:7.33-12.67months). The one-year OS and PFS rate was52%and44%,respectively. Log-rank showed that patients with low GOLPH3expression hadsignificantly longer median OS (15vs.10months in patients with high GOLPH3expression) and median PFS (12vs.7months). Univariate and multivariate analysisindicated that GOLPH3was an independent prognostic factor for OS and PFS. In the invitro experiments, GOLPH3downregulation with siRNA suppressed the proliferation,clonogenic growth and cell cycle in cultured cell lines.Conclusion: High level of GOLPH3expression in GBM tissue is associated withpoor outcome, and GOLPH3may be as a potential oncogene. Part Ⅲ: Study the relationship between the expression of BSPand the tumor garde and the prognosis of glioma patientsPurpose： Investigate the expression and prognostic value of bone sialoprotein (BSP)in glioma patients.Method: We determined the expression of BSP in normal brain and glioma samplesby immunohistochemistry using tissue microarray and real-time RT-PCR.Results: Both BSP mRNA and protein levels were significantly elevated inhigh-grade glioma tissues compared with those of normal brain and low-grade gliomatissues, and BSP expression positively correlated with tumor grade (P<0.001). In all300glioma samples, we adopted270glioma samples for analyzing (the other tissue dots werelost from the TMA slide). And in270glioma patients, we obtained258patients’ follow-up.In258glioma patients, the median overall survival (OS) was26months (95%CI:18.89-33.11months). The median progression-free survival (PFS) was21months (95%CI:16.72-25.28months). Death was found in28%LGG patients and87.3%HGG patientsduring the follow-up. Univariate and multivariate analysis showed high BSP expressionwas an independent prognostic factor for a shorter progression-free survival (PFS) andoverall survival (OS) in glioma patients [hazard ratio (HR)=1.508, P=0.014andHR=1.572, P=0.009, respectively]. Patients with low BSP expression had a significantlylonger median OS and PFS than those with high BSP expression (OS:42versus16months;PFS:30versus13months). Additionally, high-grade glioma patients who had a worseprognosis were more likely to exhibit high BSP expression (HR=2.158, PFS; HR=2.100, OS).Conclusion: High BSP expression occurs in a significant subset of high-grade gliomapatients and predicts a poorer outcome. |
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