Analysis of miR-9-5p, miR-124-3p, miR-21-5p, miR-138-5p, and miR-1-3p in Glioblastoma Cell Lines and Extracellular Vesicles.

Alja Zottel,Ivana Jovčevska,Radovan Komel,Neja Šamec,Pia Pužar Dominkuš,Lucija Raspor Raspor Dall’Olio,Daniil Nikitin,Samo Hudoklin,Maxim Sorokin,Jernej Mlakar,Ana Kump,Anton Buzdin,Rok Romih

doi:10.3390/ijms21228491

Abstract

Glioblastoma (GBM), the most common primary brain tumor, is a complex and extremely aggressive disease. Despite recent advances in molecular biology, there is a lack of biomarkers, which would improve GBM’s diagnosis, prognosis, and therapy. Here, we analyzed by qPCR the expression levels of a set of miRNAs in GBM and lower-grade glioma human tissue samples and performed a survival analysis in silico. We then determined the expression of same miRNAs and their selected target mRNAs in small extracellular vesicles (sEVs) of GBM cell lines. We showed that the expression of miR-21-5p was significantly increased in GBM tissue compared to lower-grade glioma and reference brain tissue, while miR-124-3p and miR-138-5p were overexpressed in reference brain tissue compared to GBM. We also demonstrated that miR-9-5p and miR-124-3p were overexpressed in the sEVs of GBM stem cell lines (NCH421k or NCH644, respectively) compared to the sEVs of all other GBM cell lines and astrocytes. VIM mRNA, a target of miR-124-3p and miR-138-5p, was overexpressed in the sEVs of U251 and U87 GBM cell lines compared to the sEVs of GBM stem cell line and also astrocytes. Our results suggest VIM mRNA, miR-9-5p miRNA, and miR-124-3p miRNA could serve as biomarkers of the sEVs of GBM cells.

Highlights

Glioblastoma (GBM) is the most common and highly aggressive primary brain tumor, with an annual incidence below 10 (e.g., 3.2 in the USA) per 100,000 people [1,2]
Candidate miRNAs were selected based on their target genes, vimentin (VIM), nucleolin (NCL), nucleosome assembly protein-1 like-1 (NAP1L1), FRAS1-related extracellular matrix protein 2 (FREM2), and sprouty RTK signaling antagonist 1 (SPRY1) [6,19,20,21], which were previously associated with GBM in our studies
We showed that a higher expression of miR-9-5p and miR-124-3p was related to the small extracellular vesicles (sEVs) of NCH421k or NCH644 GBM stem cell lines, respectively

Summary

Introduction

Glioblastoma (GBM) is the most common and highly aggressive primary brain tumor, with an annual incidence below 10 (e.g., 3.2 in the USA) per 100,000 people [1,2]. In Europe, the incidence varies from 3 per 100,000 people in Eastern Europe to 5 per 100,000 people in the United Kingdom and Ireland [3] This is a disease that mainly affects the elderly population, with an average age of patients diagnosed at 64 years [4]. Temozolomide is an alkylating agent that crosses the blood–brain barrier and methylates DNA, leading to cell death [7]. With this aggressive treatment, the survival of GBM patients ranges from 12 to 15 months, while only 9.8% of patients survive more than 5 years after diagnosis [8,9]. GBM stem cells are highly resistant to drugs, are migratory, and can form a new tumor [7]

Methods

Results

Discussion

Conclusion