Research On He Expression Of GOLPH3 And NDRG1 In Gliomas And Regulate Mechanism Of Apoptosis And Proliferation In Glioma Cells

Glioma is the most common adult central nervous system tumor, accounts for 44.69% in intracranial tumors. There is no doubt that we may find new treatment target and effective prevention methods by studing the risk factors and pathogenesis of glioma. Glioma is characterized by rapid cell proliferation and early transferring to adjacent normal brain tissues. It is difficult to completely remove the tumor. About the recidivation and invasion in glioma, there is still no effectively treatment options in the world.Currently, the treatment effect of gliomas is not so good,5-year survival rate is generally only about 5%.The potential cause of prognosis is rooted in its series of malignant biological behavior,including the excessive proliferation of tumor cellsx the invasion to the surrounding brain tissues, chemotherapeutic drug resistance and so on.Expecially the excessive proliferation of tumor cells, is an important factor leading to the rapid recurrence of patients with gliomas.The current treatment concept is to keep the tumor to a minimum by surgical excision under the microscope, then with radiotherapy and chemotherapy as adjuvant therapy. However, due to the special growth zone of glioma and their uncontrolled growth, the treatment effect is not good, high recurrence and mortality of glioma makes it the worst prognosis of tumors. Since the twentieth century, with the development of molecular medicine research and cancer genetics, the pathogenesis of glioma has been known more, but still didn't find an effective, specific prevention methods and therapeutic measures. So, to find a new and effective biomarkers with high specificity and sensitivity become particularly important for the early prognostic evaluation. How to effectively inhibit excessive proliferation of glioma cells, to prolong the survival time of patients and improve their quality of life, is a major problem of the current treatment of glioma. Therefore, to search the molecular mechanism of glioma cell proliferation, and do effective intervention to find therapeutic target in glioma gene therapy has become the focus of glioma research.In recent years, in order to explore the pathogenesis and susceptibility of glioma, the associated genes have been studied more and more. Specific genome and histologic characteristics of glioma make it a complex multifactorial disease with oncogenes, tumor suppressor genes and associated regulatory genes involved.Golgi phosphoprotein 3 (Golgi phosphoprotein 3, GOLPH3) is a newly discovered coat protein, unlike other membrane oncoproteins, GOLPH3 mostly lies in the Trans-Golgi Network (TGN), So it is defined as the first Golgi-oncogene. The coding gene is lacated in the 5p13 chromosome(the frequently amplified chromosomal regions in some solid tumors). Human GOLPH3 has been identified as a novel oncoprotein through genomics, clinical pathology and functional analysis. ScottKL reported in 2009 that GOLPH3 presence overexpressed in a variety of solid tumors, and participate in transport proteins, receptors circulation and glycosylation, which is required for tumor formation. Suggest that GOLPH3 proteins may be a critical role in cancer, and speculated that the role of GOLPH3 in tumor is associated with mTOR signaling pathway. The preliminary study about rapamycin sensitivity of tumor cells show that the overexpression of GOLPH3 may increase the activation of AKT. In 2009, Dippold reported that GOLPH3 is related with the normal morphology of Golgi and is beneficial to bud bubble formation. Wood reported that GOLPH3 can bind with phosphatidylinositol-4-phosphate (PtdIns(4)P), to regulate Golgi structure and function, control the secretion of vesicles within the cell, as well as anterograde and retrograde transport. These illustrated that GOLPH3 plays an important role in the formation of secretory vesicles and Golgi transport.Recent studies show GOLPH3 participates in various processes including intracellular protein transport, receptor cycling and glycosylation involved in tumorigenesis. Since first to be proved as an oncogene, GOLPH3 has been reported in many researches. GOLPH3 up-regulation is associated with poor prognosis of patients in many tumors. It maybe promote tumor cell proliferation by multiple signaling pathways, such as mTOR/AKT pathway or Wnt pathway, etc. Therefore GOLPH3 targeted drug therapy may be an effective gene target.NDRG1 (N-myc downstream regulated gene 1) was first cloned in 1997, it has been found in a variety of cancers and normal tissues, but also been found involved in embryogenesis, cell growth and differentiation, lipid synthesis, myelination, stress response and immune function. The expression of NDRG1 is regulated by multiple factors including N-myc, histone acetylation, hypoxia and the levels of calcium and iron in the cell. A number of studies have confirmed, NDRG1 can be induced in cell differentiation, the up regulation of NDRG1 will cause myelomonocytic lineage cells stop proliferation, suggesting the role of NDRG1 in the terminal differentiation. In kidney, NDRG1 can be regulated by EGR1 transcription factor through the PKD2 pathway. In addition, NDRG1 is also related with the microtubules and centrosomes, spindle esterification and a variety of P53-related genes. Down regulation of NDRG1 was related with the reduction of a-tubulin acetylation. The decrease of tubulin can affect the formation of the spindle fiber, and further affect the cell division. This study confirms the mitosis fidelity and the suppression of gene instability of NDRG1. Decreased NDRG1 expression can increase the instability of genes and further lead to tumorigenesis. Over the past decade, NDRG1 are found expressed in a variety of tumors. However, there is less research for the expression of NDRGI and the associated biological behavior in glioma, this remains to be further studied.Some scholars found that both GOLPH3 and NDRG1 are related to golgi vesicle transport. PtdIns (4) P play an important role in the structure and function of golgi. Wood reported that GOLPH3 can combine with phosphatidyl inositol-4 phosphate (PtdIns (4) P) to control the secretion of intracellular vesicles, and NDRG1 can also combine with PtdIns (4) to participate in the intracellular vesicles transport through reverse golgi apparatus ARF1. In addition, GOLPH3 and NDRG1 were both related to the formation of the cytoskeleton, spindle silk, but the correlation of GOLPH3 and NDRG1 remains to be further investigated.So, based on the research background, we first studied the expression of GOLPH3 and of NDRG1 in glioma tissues, analyzed its relationship with different glioma histological grades, and investigated the influence of GOLPH3 and NDRG1 on glioma cell apoptosis and proliferation, also discussed the potential relationship and mechanism.Part one:The study of the expression and correlation of GOLPH3 and NDRG1 in gliomas and normal brain tissueObjective In this study, the expression of GOLPH3 and NDRG1 in normal brain tissue and human glioma tissues are analyzed, and study the relationship between expression and pathological grade of glioma.Methods This experiment is divided into normal brain glioma and gliomas of grade ?, ? and ?.Normal brain tissue specimens from the patients with brain injury needs intracranial decompression surgery and no necrosis brain tissue. The expression of GOLPH3 and NDRG1 was detected in 34 primary human gliomas and 9 normal cerebral tissuses by using Real-time RT-PCR and Western blot method; and immunohistochemistry was used to examine the expression of GOLPH3 and NDRG1 protein in 76 primary human gliomas and 9 normal cerebral tissuses.Analyze the correlation between the GOLPH3 and NDRG1.Result l.The expression of GOLPH3 and NDRG1 mRNA and protein were detected in human gliomas and normal cerebral tissue. In the nontumor brain tissue, GOLPH3 showed low expression level and the expression level of GOLPH3 mRNA and protein in human glioma was significantly higher than in normal brain tissue (P <0.05),in human gliomas with increasing pathological grade increased expression,IV stage group was higher than group III level and above II level group (P<0.05).The expression level of NDRG1 mRNA and protein in human glioma II grade group was significantly higher than in normal brain tissue (P<0.05). NDRG1 mRNA and protein expression level in normal brain tissue group and III grade group, IV grade group was no statistically significant (P> 0.05). Therefore, in glioma tissues, the expression level of GOLPH3 and NDRG1 were on the contrast.2. GOLPH3 immunostaining localized to the cytoplasm in two patterns, condensed into large granules with perinuclear distribution and dispersed in the cytoplasm as fine granules.GOLPH3 positive expression rates in normal brain tissue and glioma group were 22.2%(2/9) and 57.9% (44/76). there was obvious discrepancy between two group (x=4.124, P<0.05).Grade ?, ? and IV group was pathologically identified in 39.1% (9/23),54.2%(13/24) and 75.9% (20/29),respectively.NDRG1 positive expression mainly in the cytoplasm of cells, NDRG1 in normal brain tissue and glioma group in the positive expression rate was 44.4% (4/9) and 46.1% (35/76). There was no statistical significance between the two group (x =0.008, P>0.05). Grade II, III and IV group was pathologically identified in 60.9%(14/23),41.7%(10/24) and 20.6%(7/29). respectively.In glioma tissues between immunohistochemical positive rate of GOLPH3 and NDRG1 with Western Blot detection of protein expression was the same trend. In glioma tissues, NDRG1 and GOLPH3 expression instead, and negatively correlated with both (y=-0.544, P<0.05).Conclusion I.The expression of GOLPH3 and NDRG1 have been detected in human gliomas and normal cerebral tissuses.The expression of GOLPH3 and NDRG1 may be correlation to the pathological grades of human gliomas. The expression of GOLPH3 in human gliomas was increased with increasing pathological grade. The expression of NDRG1 was decreased with increasing pathological grade in human gliomas.2. There was a negative correlation between GOLPH3 and NDRG1 in human gliomas.Part two:Research on the correlation mechanism of GOLPH3 and NDRG1 in glioma cells apoptosis and proliferationObjective To investigate the specific mechanism of GOLPH3 and NDRG1 on glioma cells'apoptosis and proliferation and exploration the relationship of GOLPH3 and NDRG1 may provide a potential candidate for the therapy of glioma.Methods 1. In cultured U87 and U251 glioma cells, the siRNAs of GOLPH3 or NDRG1 were transfected by using lipofectamine 2000, while over-expression of GOLPH3 or NDRG1 were carried by using polyjet.2. Western Blot was used to examine the protein levels; The apoptosis was analyzed by flow cytometry(FCM).CCK-8 assay were used to check the proliferation ability of glioma cells after different treatment.Results 1. The results showed that GOLPH3 siRNA-transfected U251 cells and U87 cells showed higher rate of apoptosis and the proliferation decreased compared to the cells transfected with scrambled control while NDRG1 over-expression get the same result.2. The protein level of NDRG1 and cleaved-caspase3 decreased after GOLPH3 over-expression, but increased after GOLPH3 down-regulation In U251 cells.3. NDRG1 over-expression decreased cleaved-caspase3 level but did not change the level of GOLPH3.Conclusions 1. Both of GOLPH3 and NDRG1 were correlated with apoptosis and proliferation of glioma cells. GOLPH3 and NDRG1 may provide a potential candidate for the therapy of glioma.2.knockdown of GOLPH3 promoted glioma cell apoptosis and increased the cleavage of caspase 3. In addition, we found negative correlation of GOLPH3 and NDRG1 expression in glioma tissues and provided evidence that GOLPH3 is involved in the down-regulation of NDRG1 expression in glioma cells. Down-regulation GOLPH3 promotes glioma cell apoptosis by regulating NDRG1 level and then cleaved-caspase3 activity.