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Exosome-Carried microRNA-375 Inhibits Cell Progression and Dissemination via Bcl-2 Blocking in Colon Cancer

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Florin Zaharie1,2*, Mihai-Stefan Muresan1,3*, Bobe Petrushev4, Cristian Berce5, Grigore-Aristide Gafencu6, Sonia Selicean6, Ancuta Jurj6, Roxana Cojocneanu-Petric6, Cosmin-Ioan Lisencu1,3, Laura-Ancuta Pop6, Valentina Pileczki1,6, Dan Eniu1,3, Mihai-Andrei Muresan1,3, Roxana Zaharie1,7, Ioana Berindan-Neagoe6,8, Ciprian Tomuleasa6,9, Alexandru Irimie1

1) Iuliu Hatieganu University of Medicine and Pharmacy,
2) Dept. of Surgery, Octavian Fodor Regional Institute of Gastroenterology and Hepatology;
3) Dept. of Surgical and Gynecological Oncology, Ion Chiricuta Oncology Institute;
4) Dept of Pathology, Emergency County Hospital;
5) Animal Facility, Iuliu Hatieganu University of Medicine and Pharmacy;
6) Research Center for Functional Genomics and Translational Medicine Iuliu Hatieganu University of Medicine and Pharmacy,
7) Dept. of Gastroenterology Octavian Fodor Regional Institute of Gastroenterology and Hepatology, Cluj Napoca; Romania;
8) Dept. of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
9) Dept. of Hematology, Ion Chiricuta Oncology Institute, Cluj Napoca, Romania

DOI: http://dx.doi.org/10.15403/jgld.2014.1121.244.375

Background: Worldwide, colorectal cancer (CRC) is the third most common cancer in men and second in women. The aim of the current study was to identify whether the miR-375 is indeed down-regulated in metastatic CRC and if it could be considered as a potential minimally invasive prognostic biomarker for CRC.

Methods. Exosomes were isolated and characterized from patients with liver metastasis from CCR. The characterization of exosome was performed using TEM/SEM. HCT116 cells were treated with miR-375 mimic, NSM and miR-375 inhibitor. Functional assays included cell counting assay for 14 days, Matrigel invasion assay, apoptosis assay by flow cytometry using Annexin V-FITC, RT-PCR and Western blotting.

Results. Increased proliferation potential was proven for the cells transfected with miR-375 inhibitor, while the miR-375 mimic decreased the cell number. The cells transfected with the miR-375 inhibitor are aggressive and cross the membrane; 3.84% of the cells transfected with the miR-375 inhibitor entered apoptosis, while 6.45% of those transfected with the non-specific mimic were in programmed cell death, less than those transfected with the microRNA. RT-PCR for Bcl-2 expression showed that Bcl-2 is down-regulated for miR-375 inhibitor and up-regulated for the miR-375 mimic, a result confirmed by Western blotting.

Conclusion: The present study brings to the forefront new data that suggest miR-375 as a new player in controlling the pathways responsible for inhibiting the natural history of CRC tumor cells, via the Bcl-2 pathway.

Key words: microRNA-375, colon cancer, BCL-2 pathway, biomarkers.

Abbreviations: APC: adenomatous polyposis coli; CRC: colorectal cancer; EGFR: epidermal growth factor receptor; ERB: Erythroblastic Leukemia Viral Oncogene Homolog; FITC: Fluorescein isothiocyanate; MAPK: mitogen-activated protein-kinase; miR: microRNA; mTOR: mammalian target of rapamycin; NSM: non specific mimic; qRT-PCR: quantitative real time polymerase chain reaction; SEM: scanning electron microscopy; TEM: transmission electron microscopy; TGF-beta: transforming growth factor beta.