miR-218 Affects the ECM Composition and Cell Biomechanical Properties of Glioblastoma Cells

Abstract

BackgroundGlioblastoma (GBM) is the most common malignant brain tumour. GBM cells have ability to infiltrate into the surrounding brain tissue, which results in a significant decrease in the patient’s survival rate. Infiltration is a consequence of the low adhesion and high migration of the tumour cells, two features being associated with the highly remodelled extracellular matrix (ECM). MethodsThe expression profile of miRNAs and mRNAs was analysed by qPCR on 19 GBM tissue samples. Than luciferase assay was performed to confirm interaction between miR-218 and target sequences. Next we checked how supplementation of glioma cell line (U118-MG) with microRNA 218 will change expression pattern of TN-C and SDC both on transcript level and on protein level. Analysis of protein level were made with western-blot technique. Last step in expression profiling of genes connected to cellular motility and adhesion was done with use of qPCR analysis after supplementation with miR-218. To assess the abilities of cells to migrate and proliferate real-time cell culture analysis with use of Incelligence system were utilized. Also this results were backed by classic wound-healing assay. To conclude we used atomic force microscopy (AFM) to measure physical properties such as adhesion and stiffness of cells. This study was supported by microscopy analysis of cytoskeleton changes after supplementation of miR-218.ResultsIn this study, we report that ECM composition is partially regulated at the posttranscriptional level by miRNA. Particularly, we show that miR-218, a well-known miRNA suppressor, is involved in direct regulation of ECM components, tenascin-C (TN-C) and syndecan-2 (SDC-2). We demonstrated that the overexpression of miR-218 reduces the mRNA and protein expression levels of TN-C and SDC-2, and subsequently influences biomechanical properties of GBM cells. Atomic force microscopy (AFM) and real-time migration analysis revealed that miR-218 overexpression impairs the migration potential and enhances the adhesive properties of cells. AFM analysis followed by F-actin staining demonstrated that expression level of miR-218 has an impact on cell stiffness and cytoskeletal reorganization. Global gene expression analysis showed deregulation of a number of genes involved in tumour cell motility and adhesion or ECM remodelling upon miR-218 treatment, suggesting further indirect interactions between the cells and ECM. Conclusion The results demonstrated a direct impact of miR-218 reduction in GBM tumours on the qualitative ECM content, leading to changes in the rigidity of the ECM and GBM cells being conducive to increased invasiveness of GBM.