MiR-215-5p is a tumor suppressor in colorectal cancer targeting EGFR ligand epiregulin and its transcriptional inducer HOXB9

Petra Vychytilova-Faltejskova,Jana Merhautova,Dominika Brchnelova,Regina Demlova,Jana Halamkova,José Garcia-Solano,Marek Svoboda,Ondrej Slaby,Katerina Slaba,Lenka Radova,Tana Machackova,Irene Gutierrez-Garcia,Pablo Conesa-Zamora,Igor Kiss,Rostislav Vyzula

doi:10.1038/s41389-017-0006-6

Abstract

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.

Highlights

Colorectal cancer (CRC) is the third most common cancer worldwide and the fourth leading cause of cancer related deaths
MiR-215-5p is downregulated in CRC tissues and its low levels correlate with aggressive disease It was confirmed that the expression of miR-215-5p is significantly downregulated in tumor tissue compared with adjacent mucosa (P < 0.0001; Fig. 1a) in case of Czech cohort (Table 1)
As in the Czech cohort, the expression of miR-215-5p was significantly downregulated in tumor tissues (P < 0.0001; Fig. 1b) and its low levels were associated with advanced clinical stage (P = 0.0185; Fig. 1d), but not with the lymph node positivity (Table 1)

Summary

Introduction

Colorectal cancer (CRC) is the third most common cancer worldwide and the fourth leading cause of cancer related deaths. Several miRNAs with deregulated expression in CRC have been identified, including miR215-5p11–15. We focus on miR-215-5p as we identified this miRNA to be downregulated in colorectal tumor tissue in our previous work, where it indicated promising tumor-suppressive features in preliminary in vitro functional screen. We focus on miR-215-5p as we identified this miRNA to be downregulated in colorectal tumor tissue in our previous work, where it indicated promising tumor-suppressive features in preliminary in vitro functional screen11 This miRNA is supposed to function as a tumor suppressor and its levels are often downregulated in tumor tissues. The deregulation of this miRNA is supposed to be a very early event, which is not dependent on the mechanism of initiation of transformation, suggesting that miR-215-5p is likely to regulate critical signaling pathways that are crucial for early transformation of colonic epithelial cells

Methods

Results

Conclusion