Malignant brain tumors are among the most feared types of cancer, because of their poor prognosis, as well as because of their direct repercussions on the quality of life and cognitive function . Malignant glioma is the most common type of primary malignant brain tumor accounting for 80% of patients and an annual incidence of 5.26 per 100 000 population, or 17 000 new cases diagnosed per year . Glioblastoma, as the Ⅳ-grade glioma, is the most common and most malignant tumor in glioma. Because of the limited efficacy of conventional treatments such as surgery, chemotherapy, and radiotherapy, it is urgent to study a new and effective treatment. Over the past decade, many studies have made great progress in genetics and epigenetics of GBM . Identification of relevant biomarkers for appropriate patient selection is essential for the successful development of novel therapies. In this study, we aimed to find potential biomarkers for predicting progress and prognosis in GBM.
In the present study, we obtained 716 DEGs among all three groups including 188 upregulated genes and 528 downregulated genes. These DEGs were associated with the GO gene functions involving cellular protein modification, regulation of cell communication, and regulation of signaling. Many studies reported that glioma cells can regulate cell communication and influence signaling pathway . They even can form the network, which allows multicellular communication through microtube-associated gap junctions . In addition, through KEGG pathway analysis, we found that these DEGs involved in Retrograde endocannabinoid signaling and Calcium signaling pathway, among which endocannabinoid was reported that it can act retrogradely and mediate synaptic modulation through release 2-arachidonoylglycerol (2-AG) as well as mediate long-term depression (LTD) . Furthermore, many altered calcium-binding proteins have been observed in glioblastoma multiforme, which implicated the deregulation of calcium signaling and homeostasis in GBM . In addition, glioblastoma can make patients present with epilepsy. It is known that glioma cells extrude pathological concentrations of glutamate which are thought to play a role in tumor progression and the development of epilepsy . Furthermore, the synaptic vesicle cycle involves important extracellular/endocytic processes, as well as protein complex formation/decomposition processes, which have been reported to be associated with GBM [25, 26].
We constructed PPI network to investigate the interrelationship of the DEGs, and ten hub genes were identified, including TOP2A, CDK1, BUB1, CDC20, BIRC5, MELK, KIF4A, PBK, NDC80 and TTK. All of them were upregulated genes. Their gene expression was also validated via the TCGA and Oncomine database. We conducted survival analysis for 10 hub genes. In our study, only 6 genes showed significant results, they were TOP2A, CDK1, CDC20, BIRC5, MELK and NDC80. This finding implied that the prognosis of patients with glioblastoma could be predicted by detecting the expression level of those 6 genes. Furthermore, the results of the present study provided biomarkers and targets, which may be applied in the diagnosis and treatment of patients with glioblastoma for accurate therapy.
Interesting, TOP2A, CDK1, CDC20, MELK, and NDC80 genes were all associated with the cell cycle. Cell cycle involves multiple molecular pathways that appear to be the essential mechanism of the indefinite proliferation of malignant glioma. If the gene that regulates the cell cycle progression of malignant glioma is deregulated, the development of glioma will be promoted . Most of them were reported as an essential factor involved in cell division and proliferation. In mammals, TOP2A has an important role in altering DNA topology and it is expressed in proliferating cells in late S phase, with a high in the G2 to M phases, which suggests that it has potential as a proliferation marker . Compared with lower grade astrocytomas and normal brain tissues, TOP2A transcription level in GBM patients increased significantly, which is a good prognostic indicator and can guide temozolomide chemotherapy . Cyclin-dependent kinases 1 (CDK1), located on 10q21.2, is one of the gene Cyclin-dependent kinases (CDKs) that are important regulators of cell cycle progression and cell cycle regulation . When it comes to cell proliferation, we think of the important role of CDK1 in G1/S and G2/M phase transitions, which promote the M-phase process. In addition to glioma-related, there are many cancers-related, such as lung adenocarcinoma, oral squamous cell carcinoma, etc [31–34]. CDC20, a central regulator of the cell cycle in numerous cancers, plays an essential role in the regulation of glioblastoma tumor initiating cells (TIC) proliferation, self-renewal and survival . CDC20 knockdown by transducted with shCDC20 caused loss of tumor initiating cells (TICs) in the S, M and G2cell cycle phases and accumulation in the G1 phase . In addition, silencing CDC20 expression in TIC accelerated a significant increase in apoptotic cell death . BIRC5 (Survivin) is associated with proliferation markers, histological malignancy grade, and are inversely associated with prognosis [37, 38]. Recent comprehensive studies have elaborated that knockdown of Survivin in permanent as well as primary glioma cell lines lead to immense cellular polyploidy with cells having DNA contents up to 32n, poly-merotelic kinetochore-microtubuli connections, DNA damage, DNA damage response [39–41]. Knocking down the BIRC5 in GBM cells can lead to transient G1 cell cycle arrest which was not able to halt endoreplication of DNA . MELK, paralleled with the increasing degree of malignancy in astrocytomas, is a member of the subfamily that activates the serine/threonine protein kinase . Kig C showed that siRNA-mediated loss of MELK in glioblastoma cells causes a G1/S phase cell cycle arrest accompanied by cell death or a senescence-like phenotype implied that MELK inhibitors hold great potential for the treatment of glioblastomas as such or in combination with DNA-damaging therapies .
As one of the key elements of outer kinetochore, NDC80, which has a molecular weight of 74 kDa, is a heterotetrameric protein complex that plays an important role in cell mitosis . Abnormal expression of the NDC80 causes chromosomal abnormalities, leading to instability of the genome, and genomic instability is a major factor in all tumorigenesis . Numerous studies have found that the components of the NDC80 complex are highly expressed in tumors, which can be used as a diagnostic marker for certain tumors and may even be an indicator for evaluating prognosis a. Therefore, the role of the NDC80 complex in the development of tumorigenesis has received increasing attention. However, NDC80 has rarely been reported in GBM. Our results showed the gene expression of NDC80 was upregulated in the GBM cell lines comparing the normal cells. Interestingly, the expression level of NDC80 in glioma cells (U251, U-87MG and A172) was significantly higher than that in normal astrocytes (HA1800). Moreover, its expression level is positively correlated with the degree of malignancy. These results also suggest that NDC80 plays a role in the pathogenesis and progression of GBM.