Early detection of colorectal cancer is the main prerequisite for successful treatment and reduction of mortality. Circulating microRNAs were previously identified as promising diagnostic, prognostic and predictive biomarkers. The purpose of this study was to identify serum microRNAs enabling early diagnosis and prognosis prediction of colon cancer. In total, serum samples from 427 colon cancer patients and 276 healthy donors were included in three-phase biomarker study. Large-scale microRNA expression profiling was performed using Illumina small RNA sequencing. Diagnostic and prognostic potential of identified microRNAs was validated on independent training and validation sets of samples using RT-qPCR. Fifty-four microRNAs were found to be significantly deregulated in serum of colon cancer patients compared to healthy donors (P < 0.01). A diagnostic four-microRNA signature consisting of miR-23a-3p, miR-27a-3p, miR-142-5p and miR-376c-3p was established (AUC = 0.917), distinguishing colon cancer patients from healthy donors with sensitivity of 89% and specificity of 81% (AUC = 0.922). This panel of microRNAs exhibited high diagnostic performance also when analyzed separately in colon cancer patients in early stages of the disease (T1-4N0M0; AUC = 0.877). Further, a prognostic panel based on the expression of miR-23a-3p and miR-376c-3p independent of TNM stage was established (HR 2.30; 95% CI 1.44-3.66; P < 0.0004). In summary, highly sensitive signatures of circulating microRNAs enabling non-invasive early detection and prognosis prediction of colon cancer were identified.

MicroRNAs (miRNAs) constitute a robust regulatory network with post-transcriptional regulatory efficiency for almost one half of human coding genes, including oncogenes and tumour suppressors. We determined the expression profile of 667 miRNAs in colorectal cancer (CRC) tissues and paired non-tumoural tissues and identified 42 differentially expressed miRNAs. We chose miR-215, miR-375, miR-378, miR-422a and miR-135b for further validation on an independent cohort of 125 clinically characterized CRC patients and for in vitro analyses. MiR-215, miR-375, miR-378 and miR-422a were significantly decreased, whereas miR-135b was increased in CRC tumour tissues. Levels of miR-215 and miR-422a correlated with clinical stage. MiR-135b was associated with higher pre-operative serum levels of CEA and CA19-9. In vitro analyses showed that ectopic expression of miR-215 decreases viability and migration, increases apoptosis and promotes cell cycle arrest in DLD-1 and HCT-116 colon cancer cell lines. Similarly, overexpression of miR-375 and inhibition of miR-135b led to decreased viability. Finally, restoration of miR-378, miR-422a and miR-375 inhibited G1/S transition. These findings indicate that miR-378, miR-375, miR-422a and miR-215 play an important role in CRC as tumour suppressors, whereas miR-135b functions as an oncogene; both groups of miRNA contribute to CRC pathogenesis.

Growing evidence suggests that microRNAs are involved in the development and progression of colorectal cancer (CRC). In the present study, deregulation and functioning of tumor-suppressive miR-215-5p was evaluated in CRC. In total, 448 tumor tissues and 325 paired adjacent healthy tissues collected from Czech and Spain cohorts of CRC patients have been used for miR-215-5p expression analyses. A series of in vitro experiments have been performed using transient transfection of miR-215-5p mimics into four CRC cell lines to identify specific cellular processes affected by miR-215-5p. Further, the effects of miR-215-5p on tumor growth were evaluated in vivo using NSG mice and stable cell line overexpressing miR-215-5p. Target mRNAs of miR-215-5p were tested using luciferase assay and western blot analyses. We found that miR-215-5p is significantly downregulated in tumor tissues compared with non-tumor adjacent tissues and its decreased levels correlate with the presence of lymph node metastases, tumor stage, and shorter overall survival in CRC patients. Overexpression of miR-215-5p significantly reduced proliferation, clonogenicity, and migration of CRC cells, lead to cell cycle arrest in G2/M phase and p53-dependent induction of apoptosis. The ability of miR-215-5p to inhibit tumor growth was confirmed in vivo. Finally, we confirmed epiregulin and HOXB9 to be the direct targets of miR-215-5p. As epiregulin is EGFR ligand and HOXB9 is its transcriptional inducer, we suggest that the main molecular link between miR-215-5p and CRC cells phenotypes presents the EGFR signaling pathway, which is one of the canonical pathogenic pathways in CRC.