High-grade gliomas have a complex pathogenesis, which includes mutations of many key factors of cellular signaling pathways involved in proliferation, cell migration, angiogenesis, and survival [6, 7, 10]. The study of mRNA levels of genes and their microRNAs opens up an understanding of tumor biology and new therapeutic or prognostic goals. HGGs are characterized by abnormal metabolic activity, especially in the late stages of oncogenesis [10]. The data obtained in this study indicate significant shifts in gene and miRNA expression associated with the regulation of the effector of the hypoxic response induced by hypoxia of transcription factor 1-alpha (HIF1-alpha) (Fig. 6). The level of HIF1A expression in the tumor in the study group of patients was higher than in normal brain tissue (Fig. 1, Table 2). Expression of the gene, activated by HIF1, promotes metabolic adaptation of the cell to a reduced partial pressure of oxygen and in the tumor also mediates angiogenesis and cell invasion [23].
The increase in HIF1A expression was accompanied by a decrease in the transcriptional activity of its main modulators—EGLN1 and EGLN3, respectively (Fig. 1, Table 2). The role of the products of these genes in the cell is to inactivate HIF1-alpha by hydroxylation of proline, which requires the presence of molecular oxygen [24]. In glial tumor samples, we found a decrease in the level of three miRNAs (Fig. 3) targeting HIF1A: miRNA-330-3p, miRNA-22-3p, and miRNA-107. In addition, there is a negative correlation between the expression of HIF1A and miR-122-5p, as well as EGLN1 with miR-155-5p. It is known that miR-155-5p reduces the expression of the tumor suppressor pVHL, which is involved in the degradation of HIF1-alpha and, therefore, in the cellular response to hypoxia [25].
As a transcription factor, HIF-1α is involved in the activation of the Hedgehog signaling pathway (HH/GLI) [26, 27]. HH/GLI can influence the success of GBM therapy in the case of temozolomide treatment, since the MGMT gene is one of its targets [26, 27]. Our study found an increase in the relative expression of the SMO gene (Fig. 1, Table 2), a key regulator of HH/GLI, which was accompanied by a decrease in the level of targeting for SMO and miR-324-5p (Fig. 3, Table 2).
It is known that hypoxia leads to overexpression of EGFR and promotes long-term activation of the EGFR signaling pathway [28]. In non-hypoxic conditions, EGFR activation, on the contrary, promotes the stabilization and accumulation of HIF-1α in the cell [29]. In the studied HGG samples, the average FC level of the EGFR gene was more than two times higher (Fig. 1, Table 2) compared to normal brain tissue, which was inversely proportional to the overall survival of patients (Fig. 4). The increase in the transcriptional activity of EGFR in the tumor was accompanied by a significant decrease in the expression of miRNA-122-5p, for which it is a potential target. On the other hand, there was an increase in miR-155-5p and miR-21-5p, which may also affect the expression of EGFR (Fig. 3, Table 2). Previously, Zhou X and colleagues showed that inhibition of miR-21-5p by siRNA leads to a decrease in EGFR activity [30].
According to our data (Fig. 1), the transcriptional activity of genes of the SMAD family decreased in HGG. By preventing the formation of the SMAD2/SMAD4 complex, SMAD7 is an antagonist of the TGF-β signaling pathway, which ensures the invasion and malignancy of tumor cells [31]. Recent studies strongly suggest that miRNAs act as effectors of the hypoxic response mediated by HIF1-alpha. Among the small noncoding RNAs we studied, miR-21-5p, miR-107, and miR-155-5p belong to controlled hypoxia [32]. MicroRNA-21-5p is the most typical example of overexpression in glioma tissue associated with a poor prognosis of the course of the disease [28]. It should be noted that SMAD7 is a direct target for miRNA-21-5p, which may explain the significant changes in its expression in our study.
Thus, the revealed statistically significant patterns indicate the critical role of EGFR, HIF1A, SMADs in HGG oncogenesis (Fig. 6). In the HGG tumor sample, there are both the activation of the transcription of HIF1A itself and the key participants in the associated signaling pathways EGFR, TGF-β, HIF1-alpha, HH/GLI, as well as changes in the level of microRNA (miR-215-5p, miR-122-5p, miR-21-5p, miR-326, miR-324-5p, miR-155-5p, miR-330-3p, miR-22-3p, miR-107). In addition, the altered expression pattern of HIF1A, EGFR and miRNA miR-22-3p, miR-107, and miR-330-3p is associated with decreased overall survival in HGG patients. The involvement of miRNAs in the regulation of the signaling pathways EGFR, TGF-β, HIF1-A, HH/GLI makes them attractive tools for gene therapy for oncological diseases, which currently do not have effective therapies.