The malignancy of glioblastoma depends on its ability to infiltrate adjacent tissues and to create secondary lesions [37]. The aggressive growth of glioma tumours and difficulties in developing an effective treatment scheme have led to intense integration of medical and molecular biological research. Remodelling of the ECM and miRNA deregulation are known processes contributing to GBM cell invasion and brain infiltration [38–41]. Here, we validated miR-218 as the functional regulator of ECM remodelling in a glioblastoma cell line. We found that TN-C and SDC-2 were directly regulated by miR-218, resulting in alterations in the ECM composition as well as changes in the mechanical properties of the cells. Our previous finding identified miR-218 as a potential tumour suppressor in brain tumours [33]. The sequence of miR-218 is located within intronic sequences of the SLIT2 and SLIT3 genes, and both of these genes are hypermethylated in GBM [42], which could explain its significant downregulation. Indeed, we confirmed that the expression of miR-218 is decreased by 50% in primary tumours and by ~ 70% in recurrent GBM tumours. Decreased miR-218-5p expression levels have also been reported in other types of human cancer, such as medulloblastoma, thyroid cancer and cervical cancer [43–45]. We confirmed that the predicted miR-218 targets, the ECM components TN-C and SDC-2, are directly regulated by miR-218. We used a dual-luciferase assay and miR-218 mimic to verify these functional interactions. The effects were detectable at both the mRNA and protein levels for both TN-C and SDC-2. Proteins derived from these transcripts are potentially key factors in the ECM of cancer cells [15,21]. The presence of TN-C in cancer tissues was initially considered a characteristic feature of only gliomas [46], with its expression increasing in proportion to the degree of brain tumour malignancy [47]. Its presence was found to increase the proliferation and invasiveness of cancer cells and to take part in the process of angiogenesis [48]. The role of TN-C in the neoplastic process is to reduce the adherence of cells, leading to the spread of the tumour. On the surface of healthy fibroblasts, fibronectin (FN) interacts with transmembrane proteins—integrins and syndecan-4 (SDC-4). The Rho protein is activated, and the properties of actin filaments are changed, resulting in cell adhesion. In pathological conditions, tenascin-C blocks the interaction between FN and SDC-4. The Rho protein is not activated, resulting in a lack of cell adhesion signals [49]. Considering the impact of TN-C on cancer cells and its apparent overexpression in glioblastoma tissues, it should be considered an excellent therapeutic target. Treatment with a double-stranded RNA targeting TN-C increased the average survival rate of patients [50].
An increased level of syndecan-2 is a characteristic of actively migrating cells [51]. Overexpression of this protein in melanoma cells indirectly contributes to an increase in the level of FAK kinase phosphorylation, which has a positive impact on the migration capability of these cells [17]. In lung cancer, SDC-2 deficiency prevents cells from adhering to FN, which blocks their migration [52].
ECM has become one of the most important focuses of cancer research, as it was shown to play a major role in the development of metastasis [53]. Pronounced ECM remodelling affects the invasion and migration of cancer cells [54,55]. Thus, the major regulator of cell motility is ECM stiffness. The mechanical properties of the ECM have an impact on fibronectin fibril assembly, cytoskeletal stiffness and the strength of integrin-cytoskeleton linkages, the factors found to be important for cell motility, and thus also on adhesive properties [56]. As demonstrated in previous reports, a more rigid ECM promotes glioma cell migration [57]. On highly rigid ECMs, tumour cells spread extensively, form prominent stress fibres and mature focal adhesions, and migrate rapidly [57]. Our results are in line with these observations, as we showed a decreased cell migration rate after mir-218 overexpression, with subsequent downregulation of TN-C expression. These direct effects were enhanced by the indirect effect of miR-218 on a number of proteins, e.g., fibronectin, collagens or laminins. Thus, with miR-218 overexpression, we observed changes in the ECM leading to slowed cell migration, most likely induced by changes in overall ECM rigidity.
Together with the cell volume and cytoskeletal dynamics, the ECM composition is another important parameter that influences the “Go-or-Grow” phenomenon of glioma cells [58]. The “Go-or-Grow” decision is strictly regulated and modulated by changes in the tumour microenvironment, which allows cells to “Go” towards more favourable conditions to proliferate at the distant site or to “Grow” if their current environment provides the proper conditions for tumour growth. Changes in miRNA expression can modulate the “Go-or-Grow” decision. Overexpression of miR-451 was shown to be related to shorter survival times of GBM patients and to increased cell proliferation [59]. In contrast, the considerable overexpression of miR-9 in GBM was shown to inhibit proliferation but concurrently promote migration. These results are consistent with our observations, where we found that miR-218 leads to a decrease in the migration rate. In addition, we observed an increase in the proliferation potential (Fig. 4). This finding could then suggest that changes in the ECM properties lead to changes in cell migration behaviour impacted by the more favourable environment promoting cells to stay at the site of origin. There is evidence indicating that mechanical properties and deformability can be used as biomarkers to distinguish between healthy and cancer cells. The deformability of a whole cell, which depends on the properties of the cytoplasm, the cytoskeleton and the nucleus, can be defined in terms of the response of the cell to an applied stress. One of the techniques that enables the measurement of biophysical properties of cells, such as adhesion and stiffness, is AFM [60]. We evaluated the mechanobiological properties of GBM cells, including adhesion and stiffness, upon miR-218 mimic treatment. We obtained real-time measurements in cell culture (xCELLigence system) and measured physical forces and the work of adhesion [61] by application of AFM in SCFS mode. This approach allowed us to quantify the adhesion of single cells. SCFS analysis revealed strengthened adhesion of GBM cells upon miR-218 overexpression, hence indicating the direct connection between miR-218 and ECM component regulation.
GBM cells, similar to other solid cancers, can remodel the surrounding microenvironment from a normal brain to a stiffer tumour microenvironment through the combination of proteolytic degradation of some ECM components and secretion of other novel ECM components [62]. In our analysis, the stiffness of miR-218-transfected cells as measured by AFM was 30% higher compared to the control cells. Despite the variability observed in the experiment, a clear difference was observed, as the overall stiffness was measured to increase in cells treated with miR-218. The differences observed in the experimental cell group might have stemmed from the distributed contribution of surface receptors on an individual single cell, which can thus impact the adhesion of that cell [63,64]. It has already been shown that tumours can become stiffer than normal tissues due to increased Rho-dependent cytoskeletal pressure, generating excessive growth, focal adhesions, adjacent joint division, and tissue disruption [65]. Stiffness also directly depends on the malignancy of the tumour. It is known that invasive GBM tumours produce stiffness-promoting factors such as collagen, fibronectin and laminins, which may suggest that the production of these proteins is disrupted after miR-218 overexpression [66].
An increase in stiffness has also been observed in many different types of cancer cells, such as breast cancer, melanoma, prostate cancer and cervical cancer cells. An important aspect of cell stiffness is the ratio of cancer to normal cells. While cancer cells are less stiff than normal cells [67], the same pattern of stiffness is also observed in malignant versus non-malignant tissues in breast cancer [68], bladder cancer [69] and prostate cancer [70]. In our research, glioblastoma cells with miR-218 overexpression were approximately 30% stiffer than non-treated cells. Increased stiffness in brain tissues can be correlated with diseases such as brain abscess or with cytoskeletal maturation in brain cells [71]. The correlation of cytoskeletal maturation with an increase in cell stiffness has been observed for astrocytes, in which the AFM-measured stiffness may increase sevenfold in a 5-week observation period during development [72]. In miR-218-treated GBM cells, the actin cytoskeleton was slightly rearranged, which could explain the increase in cell stiffness.
The minor discrepancy in the relation between cell surface adhesive properties and cell stiffness measured in our study can be explained by the different scales of measurements. For cell adhesion, SCFS measurements are limited to local changes occurring on the cell surface, while the stiffness reflects the overall mechanical properties of cells. Thus, we analysed stiffness assuming an indentation depth of 200 nm. This value assures the sensing of a superficial layer of actin filaments. Additionally, brain tissue is much softer than other tissues. The value of Young’s modulus ranges from 1 to 1,9 kPa for white matter and from 0,8 to 1,4 kPa for grey matter, depending on the measurement technique [73,74]. Independent methods, i.e., SCFS and the xCELLigence system, showed similar increases in the adhesive properties of U-118 MG cells upon miR-218 treatment. Collectively, these results demonstrated that miRNA-218 strongly affects the expression of genes encoding cell surface receptors responsible for the adhesive properties of cells.
We also found that miR-218-treated cells are more rigid than non-treated cells, which most likely prevents them from undergoing extravasation and intravasation during migration and invasion events. We thus hypothesized that miR-218 overexpression supports the maintenance of the normal-like cell phenotype, which is correlated with differences in mechanical properties. The observation is more important when one realizes the importance of ECM rigidity in the perivascular space. It has already been shown that this part of the brain tissue is more rigid in GBM, thus promoting glioma cell migration [57].
miR-218-5p deregulation is involved in GBM growth and migration potential. In addition to the direct influence that miR-218 has on transcripts such as TN-C or SDC-2, as shown in this study, it can influence the ECM composition by targeting other molecules, e.g., the Wnt/β-catenin pathway transcription factors LEF1 or MMP-9 [75]. There are data showing that miR-218 suppresses cell invasion and spheroid formation [76], arrests GBM cells in G1 phase [31] and can reduce the expression of cancer stem cell markers such as CD133, SOX2 and Nestin [77]. The complex influence that miR-218 has on GBM cells cannot be underestimated and studied only by evaluating direct targets of this miRNA, therefore in search of indirect targets of miR-218 in glioblastoma, we performed an extended expression analysis and found 47 genes connected to focal adhesion and cell motility. After miR-218 overexpression in glioblastoma cells, we observed a decrease in the expression levels of GBM oncogenes such as PIK3CA, ROCK1, LAMC1, and ICAM1. The expression of these genes is increased in GBM compared to healthy tissues [78–81]. Enhanced miR-218 levels also reduced the expression levels of the CRK, RHOA and PTPN1 genes involved in GBM progression [82–84]. Our results are supported by data in the literature indicating a decrease in the expression levels of PIK3CA [85], RHOA [86] and STAT3 [45,87] as a consequence of miR-218 overexpression.
Due to the nature of our research, the changes in the expression levels of CDC42, STAT3, EGF and CTTN might be particularly important. Previous reports have indicated that CDC42 is a critical determinant of the migratory and invasive phenotype of malignant gliomas [88,89]. The STAT3 level is correlated with GBM malignancy, indicating its participation in increasing the migration potential of cancer cells [90]. Additionally, regarding EGF, its impact on the migratory nature of GBM cells is known [91]. CTNN and the Arp2/3 complex are known for regulating lamellipodia formation, and a decrease in CTNN expression can suppress GBM migration mechanisms [92,93]. Because we showed a decrease in the migration capacity of glioblastoma cells under treatment with miR-218 in our studies, we can conclude that these changes are the result of the impact of miR-218 on CDC42, STAT3, EGF and CTTN.
The observed increase in GBM cell adhesion may also be associated with a decrease in ACTN1 expression. It has been shown that after downregulation of ACTN1, GBM cells show poor spread but increased focal adhesion [94]. The changes in the cytoskeleton that we observed may be the result of a reduced HGF level, which has been demonstrated to affect the distribution of the actin cytoskeleton in glioblastoma cell lines [95]. Both the cancer migration pathway and deregulation of the actin cytoskeleton can be related to downregulation of SH3PXD2A after miR-218 overexpression. SH3PXD2A is a crucial element in the formation of actin-based invadopodia–protrusions of the plasma membrane that are associated with mechanisms of invasiveness [96,97].