In silico studies are essential to finding new pathways and genes involved in biological processes. This study used the GSE32466 data set, which includes both recurrent and primary GBM groups. GBM angiogenesis plays an essential role in its recurrence, and the GSE32466 data set is the best available dataset containing data on GBM recurrence[3]. Our main goal was to identify classes of indirect pathways that induce VEGF in GBM recurrence. Further understanding of GBM neoangiogenesis may improve our understanding about mechanisms of resistance to anti-angiogenetic drugs. The development of multi-omics approaches has led to an impressive deal of new information on a variety of mechanisms and created the opportunity to target GBM selectively [9]. In this study, by identifying the miR-DEGs, we found the pathways they target. These pathways seem to be involved in the recurrence of GBM and may also include pathways associated with angiogenesis. The most well-known angiogenic pathway currently treated in cancer therapy is the VEGF signaling pathway. Therefore, we chose the VEGF pathway as our focus hoping that by simultaneously targeting multiple pathways affecting angiogenesis, we may improve the chances of effectively controlling angiogenesis in cancer therapy [15]. Studies showed that, there are other signaling pathways affecting the function of the VEGF pathway. For example, a study has shown that glioma cells produce TGF-β superfamily ligands which bind TGF-β receptors (TGF-βR). In other study, Sui et al. found that VEGF-mediated angiogenesis was activated in the normoxia mode by the TGF-β1 / β-catenin / TCF3 / LEF1 signaling pathway, and in the hypoxia by the HIF-1α / β-catenin / TCF3 / LEF1 signaling pathway [16 17]. Therefore, the TGF-β pathway can be one of the pathways affecting the function of the VEGF pathway.
Other studies have shown that the HGF/c-MET signaling pathway plays an important role in angiogenesis and tumor growth. Hence, the function of this signaling pathway is synergistic with the VEGF/VEGFR signaling pathway, for instance they have a greater stimulating effect on angiogenesis and tumor growth when treated simultaneously [18, 19]. Accordingly, the purpose of our study was in silico identification of the pathways with the potential to target the VEGF signaling pathway in recurrent GBM indirectly. In this study, the pathways with overlapping genes or pathways that indirectly affect the VEGF signaling pathway affecting GBM recurrence, were investigated. The VEGF/R targeting therapies are designed to treat GBM angiogenesis; however, the clinical effect of these therapies is limited due to the emergence of drug resistance during treatment [4]. The mechanisms underlying cancer drug resistance is still not fully understood. Therefore, it is critical to discover the mechanisms of drug resistance to increase the success rate of GBM treatment, which is a recurrent disease [20, 21]. In recurrent GBM, chromosomal instability (CIN) is considered an important mechanism leading to tumor heterogeneity. In addition, high heterogeneity affects the disregulation of microRNA levels and, consequently, their related functions [9]. In this study, we reached the effective pathways through DEmiRNAs in recurring GBM. Based on our findings, among 79 significant DEmiRNAs, eight miRNAs overlapped with the VEGF pathway. Therefore, it seems that if other microRNAs play any role in angiogenesis, they may also indirectly affect the VEGF pathway. Indirect effects may occur in the form of connection or overlap, both of which were examined using NetworkAnalyst. Zhang et al. found that, the anticancer drug convallatoxin induces apoptosis in colon cancer cells by cross-linking the JAK2/STAT3 and mTOR/STAT3 signaling pathways and inhibiting angiogenesis [22]. Bo Zhang et al. showed that increasing LncRNA ANRIL expression caused the overexpression of the VEGF gene and induced angiogenesis by activating the NF-κB signaling pathway in a mouse model of diabetes mellitus with stroke [23]. Another study found that activation of the Jagged1/Notch1 signaling pathway by 17β-esteradiol is associated with angiogenic factors that may turn tumor cells into angiogenic phenotypes [24]. In the study by Nabors et al., it was shown that, extracellular signals such as hypoxia and cytokines induced over-expression of angiogenic factors in glioma cells [25].
GBM cells are involved in many mutations that significantly affect cancerous processes. These mutations lead to low survival rates by increasing the complexity of treatment and resistance to therapy in patients. Different pathways in the intracellular signaling network are dis-regulated due to mutations [26, 27]. Our study and others also showed that signal transduction pathways are the main pathways that affect angiogenesis. In a functional study in a breast cancer cell line, Zhang et al. showed that the MAPK signaling pathway plays a role in regulating angiogenesis. In this pathway, HGF/SF, together with its receptor (c-MET), causes angiogenesis by increasing VEGF gene expression [28]. Twelve percent of the pathways identified and examined in our study affect other cancers as well. In our study, various endocrine systems were identified as pivotal pathways indirectly related to the VEGF pathway. Various studies have emphasized the role and impact of these pathways on the patients, survival, cancer development, and migration of cancer cells and the recurrence of GBM, thus affecting GBM patient survival rates [29, 30]. Consistent with this study, the role of steroid hormones, prolactin (PRL) and its receptor (PRLR), and leptin-related signaling in GBM has been identified in some functional studies, making them potential therapeutic targets for future studies [31–33]. The role of oncoviruses in various cancers has also been identified. There are not many studies on the role of viruses in GBM, but McFaline-Figueroa et al., has pointed to the presence of cytomegalovirus in GBM [34]. Another class of pathway identified in our in silico analysis is the eukaryotic community. This class includes signaling pathways mostly associated with invasion and apoptosis in cancers, including those in GBM [35].
Interestingly, some of the final pathways we identified are shared between GBM and some diseases and processes not related to cancer. Some of these pathways are associated with neurodegenerative disease, cardiovascular disease, endocrine and metabolic disease, and the digestive system. Perhaps the reason for their apparent irrelevance is the absence of studies investigating the correlation or the common factors between them and GBM. However, in some neurodegenerative diseases, such as prions, a link between the prion protein (PrPC) and GBM has been observed [36]. The immune system is another class identified in our study. GBM has mechanisms that can help it escape the central nervous system (CNS) immune system and promote some of its own processes, such as angiogenesis [37, 38]. Some treatments are also being considered in the relapse phase; folding, sorting, degradation, and transport are processes that have not received much attention in cancer studies. However, one study has suggested the role of UBE3C in ANXA7 ubiquitination in the progression of glioma [39]. Other critical pathways requiring further laboratory studies are those associated with beta-adrenergic, cytoskeletal, endocrine, and metabolic diseases. The extracellular matrix (ECM) in GBM is a unique environment that plays an essential role in creating the invasion phenotype and GBM recurrence [40]. Some lipid metabolism is related to some properties of GBM, including those involved in the maintenance of cancer stem cells (CSCs) [41]. The pathways identified in our analysis may be related to both angiogenesis and the recurrence of GBM.
In GBM, 51 genes are altered in the VEGF signaling pathway (Table 1). However, according to our in silico results, there are other signaling pathways that can indirectly affect VEGF signaling pathway genes expression levels.