Differential gene expression of immunity and inflammation genes in colorectal cancer using targeted RNA sequencing

Veronika Holubekova; Dusan Loderer; Marian Grendar; Peter Mikolajcik; Zuzana Kolkova; Eva Turyova; Eva Kudelova; Michal Kalman; Juraj Marcinek; Juraj Miklusica; Ludovit Laca; Zora Lasabova

doi:10.3389/fonc.2023.1206482

Differential gene expression of immunity and inflammation genes in colorectal cancer using targeted RNA sequencing

Front Oncol. 2023 Oct 5:13:1206482. doi: 10.3389/fonc.2023.1206482. eCollection 2023.

Authors

Affiliations

¹ Laboratory of Genomics and Prenatal Diagnostics, Biomedical Center in Martin, Jessenius Faculty of Medicine, Comenius University in Bratislava, Martin, Slovakia.
² Laboratory of Bioinformatics and Biostatistics, Biomedical Center in Martin, Jessenius Faculty of Medicine, Comenius University in Bratislava, Martin, Slovakia.
³ Clinic of Surgery and Transplant Center, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin University Hospital, Martin, Slovakia.
⁴ Department of Molecular Biology and Genomics, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia.
⁵ Department of Pathological Anatomy, Jessenius Faculty of Medicine, Comenius University in Bratislava, Martin University Hospital, Martin, Slovakia.

Abstract

Introduction: Colorectal cancer (CRC) is a heterogeneous disease caused by molecular changes, as driver mutations, gene methylations, etc., and influenced by tumor microenvironment (TME) pervaded with immune cells with both pro- and anti-tumor effects. The studying of interactions between the immune system (IS) and the TME is important for developing effective immunotherapeutic strategies for CRC. In our study, we focused on the analysis of expression profiles of inflammatory and immune-relevant genes to identify aberrant signaling pathways included in carcinogenesis, metastatic potential of tumors, and association of Kirsten rat sarcoma virus (KRAS) gene mutation.

Methods: A total of 91 patients were enrolled in the study. Using NGS, differential gene expression analysis of 11 tumor samples and 11 matching non-tumor controls was carried out by applying a targeted RNA panel for inflammation and immunity genes containing 475 target genes. The obtained data were evaluated by the CLC Genomics Workbench and R library. The significantly differentially expressed genes (DEGs) were analyzed in Reactome GSA software, and some selected DEGs were used for real-time PCR validation.

Results: After prioritization, the most significant differences in gene expression were shown by the genes TNFRSF4, IRF7, IL6R, NR3CI, EIF2AK2, MIF, CCL5, TNFSF10, CCL20, CXCL11, RIPK2, and BLNK. Validation analyses on 91 samples showed a correlation between RNA-seq data and qPCR for TNFSF10, RIPK2, and BLNK gene expression. The top differently regulated signaling pathways between the studied groups (cancer vs. control, metastatic vs. primary CRC and KRAS positive and negative CRC) belong to immune system, signal transduction, disease, gene expression, DNA repair, and programmed cell death.

Conclusion: Analyzed data suggest the changes at more levels of CRC carcinogenesis, including surface receptors of epithelial or immune cells, its signal transduction pathways, programmed cell death modifications, alterations in DNA repair machinery, and cell cycle control leading to uncontrolled proliferation. This study indicates only basic molecular pathways that enabled the formation of metastatic cancer stem cells and may contribute to clarifying the function of the IS in the TME of CRC. A precise identification of signaling pathways responsible for CRC may help in the selection of personalized pharmacological treatment.

Keywords: KRAS mutation; RNA sequencing; colorectal cancer; differentially expressed genes (DEG); immune system; inflammation; signaling pathways.

Grants and funding

This work was supported by grants from The Ministry of Education, Science, Research and Sport of the Slovak Republic (grant VEGA no. 1/0269/22) and by The Slovak Research and Development Agency (grant APVV no. 16-0066 and no. 21-0448), and by the European cooperation in science and technology no. COST CA17118.