Sequencing of long non-coding RNAs in exosomes of colorectal cancer patients

Czech version

Authors: M. Madrzyk ^1,2; T. Macháčková ¹; K. Trachtová ¹; T. Catela Ivković ¹; K. Součková ¹; J. Kotouček ³; J. Mašek ³; T. Loja ¹; M. Šachlová ⁴; O. Slabý ¹
Authors‘ workplace: CEITEC – Středoevropský technologický institut, MU Brno ¹; LF MU Brno ²; Výzkumný ústav veterinárního lékařství, v. v. i., Brno ³; Gastroenterologické oddělení, MOÚ Brno ⁴
Published in: Klin Onkol 2022; 35(Supplementum 1): 138-141
Category: Article

Overview

Background: The prognosis of patients with colorectal cancer (CRC) depends mainly on the extent of the disease at the time of diagnosis; therefore, early detection is one of the main prerequisites for successful treatment. Current research shows that exosomal long non-coding RNAs (lncRNAs) are associated with cancer development. As lncRNAs are often tissue specific, their quantification in exosomes is proposed as a non-invasive method for early detection of CRC. In this study, we aimed to optimize a protocol for analyzing exosomal lncRNAs from blood serum of CRC patients as potential diagnostic biomarkers. Material and methods: Exosomes were isolated by gel chromatography from 150 μl of serum of CRC patients and healthy donors. Their quality and quantity were confirmed by electron microscopy and dynamic light scattering (DLS) analysis; protein markers were detected by Western blot. After RNA isolation, cDNA libraries were prepared and sequenced using NextSeq 550. Results: We successfully isolated exosomes and verified them by several methods. Libraries were prepared from all samples despite very low volume of starting material. The sequencing data confirmed the presence of both protein-coding (50%) and non-coding RNAs, which consisted mainly of lncRNAs (28.2%), pseudogenes (15.2%) and other RNA types (6.5%). The results also showed significantly altered levels of some lncRNAs that could distinguish samples from CRC patients and healthy controls. Using gene set enrichment analysis (GSEA), we observed significantly enriched classes of genes related to DNA repair or cell cycle regulation. Conclusion: Our preliminary data suggest that lncRNAs represent a significant fraction of the RNA present in exosomes and that their distinct levels can separate CRC patients from healthy controls. The analysis of enriched genes also showed a significant representation of lncRNAs involved in cell cycle regulation and DNA repair, suggesting their possible involvement in cancerogenesis. However, the results need to be verified in a larger cohort of patients.

Keywords:

gene expression profiling – colorectal neoplasms – Exosomes – RNA, long noncoding – biomarkers, tumor

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