| Backgrounds and Objectives:Gliomas are the most common primary neoplasms in the central nervous system and account for 80% of malignant primary tumors of brain. World Health Organization (WHO) classification divides gliomas into 4 grades with increasing degree of malignancy. Glioblastoma (GBM) belonging to WHO IV constitutes the majority of gliomas and is the most challenging forms to treat. Currently, only surgical resection and adjuvant chemotherapy with temozolomide combined with radiotherapy are standard-of-care treatment strategies for this disease. However, the malignant behavior of these cancers with resistance to chemotherapy and radiation results in a high recurrence rate, and thus patients with malignant glioma derive little benefit from standard treatments. The disease ultimately follows a fatal course with the median survival of 12 to 15 months and 2 to 5 years for patients with GBM and anaplastic glioma, respectively.Due to the development of molecular biology and genomics, more and more genomic events and molecular mechanisms involved in tumor development have been found. Identification and targeting vital components in core signal pathways which suppress activation of receptors and signal transduction will inhibit angiogenesis and tumor growth and induce apoptosis. Elucidation of the molecular pathology of gliomagenesis and maintainance of the malignant phenotypes undoubtedly offers support to the realization of molecular targeted therapy for cancer patients.Advent of high-throughput will open opportunities to rationally develop effective molecular targeted therapeutic strategies. Combined with complete follow-up data, therapeutic response and survival differences, the usage of this technology such as next-generation of sequencing, gene microarray and tissue microarray can facilitate us to analyze changes of tumor in gene and molecule levels, uncover differently expressed genes, and find reliable therapeutic targets. The Cancer Genome Atlas (TCGA) project led by the National Cancer Institute (NCI) and National Human Genome Research Institute (NHGRI) initiated in 2006 aims to strengthen the understanding of molecular mechanism of tumor via high-throughput technology. Studies of GBM have been the focus of the project. With clustering analysis, analysis of gene differential expression and survival analysis, TCGA gene expression data combined with clinical information of GBMs and data of glioma tissues underwent gene microarray will be incorporated and genes abnormally expressed in glioma and related to prognosis will be found. Results of the data analysis not only help us to understand the molecular pathogenesis of glioma, but also reveal some useful molecular markers for prognosis evaluation in glioma patients, which all eventually lay a potent foundation for the realization of individual therapy.GPR65 belonging to the family of GPCRs acts as an acid sensing receptor. Given that malignant tumors can undergo the starvation of glucose and chronic hypoxia due to the highly rapid proliferation, which can result in lactic acid stack and low extracellular pH, however, cancer cells can adapt to the stresses to survive. Previous studies have demonstrated that under the acidic condition, GPR65 can be internalized from the plasma membrane, then activates several signaling pathways to improve cell proliferation and protect against acidosis-related cell injury. Till now, there haven’t been any reports about GPR65 in gliomas. Here, we comprehensively analyze data from the database TCGA, gene microarray and tissue microarray, try to find the effect of GPR65 on glioma patients’ prognosis, and study the role of GPR65 in proliferation of malignant glioma cell with in vitro experiments.Methods and Results:Previously, we have collected 15 glioma samples which all met pathologic diagnostic criteria and included one glioma of grade WHO Ⅰ, seven gliomas of WHO Ⅱ, two gliomas of WHO Ⅲ and five gliomas of WHO IV. Then these glioma tissues were cracked into fragments, from which RNA was extracted and inversed into cDNA that would be made into gene microarray chip. Signal of gene was obtained by a scanner with the method called sequencing by hybridization. Gene signals of two groups, which were defined by patients’status at the end of follow-up, were calculated by t-test and fold-change. Time of the follow-up of all 15 glioma patients ranged from 5 months to 85 months. By the end of the follow-up, seven patients had been dead and eight patients had been still alive. With heat-map analysis, we found that 134 genes could distinguish the two groups of different status at the level of p<0.015 and fold-change> 2. We downloaded TCGA gene expression data combined with clinical information of more than 500 GBM patients. These 134 genes were validated one by one in TCGA dataset with gene expression profile and clinical information of 528 GBMs and 10 normal brain tissues by rank sun test, Kaplan-Meier survival analysis and log-rank test. GPR65 was found out that it was overexpressed in GBM and related to patients’prognosis. Overall survival of GBM patients with high GPR65 expression were significantly shorter that of patients with low GPR65 expression (median OS 11.7 months vs.14.2 months, p=0.003).Then, we collected 300 glioma tissues (11 gliomas of WHO Ⅰ,109 gliomas of WHO Ⅱ,47 gliomas of WHO Ⅲ and 133 gliomas of WHO IV) and 16 normal brain tissues. During the long-term follow-up, complete data of 285 patients was obtained. Till the last follow-up by September 2013,39.3% of all patients had been alive and tumor recurrence was not found by imaging in 35.4%. The median OS and PFS of all patients were 30 and 26 months, respectively. We utilized high-throughput tissue microarray and immunohistochemical (IHC) methods to verify the expression level of GPR65 in glioma and normal brain tissues and its relationship with prognosis of glioma patients. Result of IHC staining of 281 glioma samples and 16 controls revealed that GPR65 expression was markedly elevated in gliomas compared with that of normal brain tissues (p<0.001). Kaplan-Meier analysis showed that median overall survival of 117 glioma patients with high GPR65 expression was statistically shorter than that of 164 patients with low GPR65 expression (OS:15 vs.34 months, p<0.001; PFS:12 vs.30 months, p<0.001). Furthermore, subgroup analysis also displayed positive result that high GPR65 expression was related to short median survival in primary GBM patients (OS:11 vs.14 months, p=0.01; PFS:8 vs. 11 months, p=0.022). Multivariate analysis revealed that high expression of GPR65 could be an independent risk factor for overall survival (Hazard Ratio=1.599,95%CI 1.028-2.487, p=0.037) and progression-free survival (Hazard Ratio=1.593,95%CI 1.036-2.450, p=0.034) of primary GBM patients.To further investigate the function of GPR65 in the tumorgenesis of glioma, we firstly constructed and identified the lentiviral-mediated RNA interference and overexpression system in glioma cell line U251 with quantitative PCR and Western-blot. Then cell counting kit-8 (CCK-8), clonoy formation and flow cytometry were applied to assess the proliferation rate, clonogenic ability and cell cycle changes of the transfected cell, respectively. We observed that upregulated GPR65 did really facilitate the growth and clonogenic ability of glioma cell compared with negative control, in contrast, interference of GPR65 expression resulted in inhibit tumor cell proliferation, clonoy formation and arrested the cell cycle in G2 phase.Conclusion:In summary, our research demonstrated that GPR65 was overexpressed in gliomas and could predict glioma patient’s survival based on data analysis of public database, gene microarray and tissue microarray of clinical tissue samples. In primary GBM patients, GPR65 would act as an independent prognostic factor. Moreover, we also found GPR65 enhanced proliferation of glioma cell. All indicates that GPR65 can be a candidate target in the further development of molecular targeted therapy for glioma patients. |
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