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Open AccessArticle

Validation of miR-1228-3p as Housekeeping for MicroRNA Analysis in Liquid Biopsies from Colorectal Cancer Patients

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Gastrointestinal & Pancreatic Oncology Group, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD) /Hospital Clínic of Barcelona/Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, 08036 Barcelona, Catalonia, Spain
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Bioinformatics Platform, CIBEREHD, 08036 Barcelona, Catalonia, Spain
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Department of Gastroenterology, Complexo Hospitalario Universitario de Ourense, Instituto de Investigación Sanitaria Galicia Sur, CIBEREHD, 32005 Ourense, Spain
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Department of Gastroenterology, Hospital Universitario de Burgos, 09006 Burgos, Spain
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Department of Gastroenterology, Hospital Donostia/Instituto Biodonostia, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD). Universidad del País Vasco UPV/EHU, 20014 San Sebastián, Spain
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Department of Gastroenterology, Hospital Clínico Universitario, IIS Aragón, University of Zaragoza, CIBEREHD, 50009 Zaragoza, Spain
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Gastroenterology Unit, Hospital General Universitario de Alicante, 03010 Alicante, Spain
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Department of Gastroenterology, Complexo Hospitalario Universitario de Vigo, 36214 Vigo, Spain
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Department of Gastroenterology, Hospital Universitario de Canarias, Universidad de La Laguna, Instituto Universitario de Tecnologías Biomédicas (ITB) & Centro de Investigación Biomédica de Canarias (CIBICAN), 38320 San Cristobal de La Laguna, Tenerife, Spain
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Advanced Marker Discovery (AMADIX), Acera de Recoletos 2, 47004 Valladolid, Spain
*
Author to whom correspondence should be addressed.
Biomolecules 2020, 10(1), 16; https://doi.org/10.3390/biom10010016
Received: 12 November 2019 / Revised: 16 December 2019 / Accepted: 18 December 2019 / Published: 20 December 2019
(This article belongs to the Special Issue microRNA Biomarkers in Clinical Study)
Background: Circulating microRNA (miRNA) analysis is a growing research field. However, it usually requires an endogenous control or housekeeping (HK) in order to normalize expression of specific miRNAs throughout different samples. Unfortunately, no adequate HK for circulating miRNA analysis is still known in the colorectal cancer (CRC) context whereas several have been suggested. Hence, our aims were to validate the previously suggested miR-1228-3p as HK for CRC studies, to compare its suitability with the widely used miR-16-5p, and to evaluate the influence of hemolysis on both miRNAs. Methods: We analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) the expression of miR-1228-3p, miR-16-5p and the spike-in cel-miR-39 in a set of 297 plasmas (92 CRC, 101 advanced adenomas -AA-, and 100 controls) and 213 serum samples (59 CRC, 74 AA and 80 controls). We also analyzed both miRNAs depending on the hemolysis degree in 7 plasmas and 31 serums. Results: Levels of miR-1228-3p and miR-16-5p did not show significant differences between groups although miR-16-5p exhibited more variability in plasma and serum samples. Importantly, the combination of cel-miR-39 and miR-1228-3p was the most stable one. Moreover, we observed that miR-16-5p was significantly influenced by hemolysis in contrast with miR-1228-3p that exhibited no correlation with this confounding factor in both biofluids. Conclusion: MiR-1228-3p has been validated as an adequate endogenous control for circulating miRNA analysis in CRC and AA liquid biopsies. View Full-Text
Keywords: biomarker; tumor marker; plasma; serum; colon cancer; qRT-PCR; reference miRNA; endogenous control; normalization; non-invasive diagnosis biomarker; tumor marker; plasma; serum; colon cancer; qRT-PCR; reference miRNA; endogenous control; normalization; non-invasive diagnosis
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Duran-Sanchon, S.; Vila-Navarro, E.; Marcuello, M.; Lozano, J.J.; Muñoz, J.; Cubiella, J.; Diez, M.S.; Bujanda, L.; Lanas, A.; Jover, R.; Hernández, V.; Quintero, E.; Herreros-Villanueva, M.; Martín, A.C.; Pérez-Palacios, R.; Arroyo, R.; Castells, A.; Gironella, M. Validation of miR-1228-3p as Housekeeping for MicroRNA Analysis in Liquid Biopsies from Colorectal Cancer Patients. Biomolecules 2020, 10, 16.

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