Original ResearchBasic and Translational—Alimentary TractStrand-Specific miR-28-5p and miR-28-3p Have Distinct Effects in Colorectal Cancer Cells
Section snippets
Colorectal Samples
Eighty-five CRC samples and 26 normal colorectal tissue samples (of which 24 were paired) were collected between 2003 and 2008 at the University Hospital of Ferrara in Ferrara, Italy (first sample set). Forty-two tumors were classified as microsatellite stable (MSS), and 43 tumors were classified as microsatellite unstable (MSI) (Supplementary Methods). For a confirmation set of samples, we obtained 23 paired samples of tumor and adjacent colorectal tissue that were collected between 2002 and
miR-28-5p and miR-28-3p Are Down-regulated in CRC
Expression levels of miR-28-5p and miR-28-3p were analyzed by quantitative real-time polymerase chain reaction (PCR) in 85 human CRC specimens and 26 normal human colorectal specimens. In order to ensure that the reference gene snRNA U6 does not change between normal and tumor samples, we calculated the mean Ct values as 2−Ct. Levels of U6 did not differ between normal and tumor tissue, 2−CtTumor/2−CtNormal = 0.94 (P = .41) (Supplementary Figure 2). Both miRNA-28-5p and miR-28-3p were
Discussion
In the present study, we analyzed 2 independent sets of human CRC samples, for a total of 108 (47 paired with normal tissue), and found significant down-regulation of both mature miR-28 forms. Our study is the first to show down-regulation of miR-28 in cancer. In the literature, only 1 study extensively analyzed miR-28 function in cancer, namely in myeloproliferative neoplasms. Girardot et al identified miR-28 overexpression in platelets of BCR-ABL–negative myeloproliferative neoplasm patients
Acknowledgments
The authors thank Sue Moreau from the Department of Scientific Publications at The University of Texas MD Anderson Cancer Center for English language editing of the manuscript. The authors also thank Dr Thomas Schmittgen from Ohio State University, Columbus OH, and Dr Ramiro Magno and Dr Stan Marée from John Innes Center, United Kingdom, for technical advice on qRT-PCR data analyses.
Drs Nicoloso and Spizzo are currently at the Division of Experimental Oncology, CRO, National Cancer Institute,
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Conflicts of interest The authors disclose no conflicts.
Funding M.I.A. is supported by a PhD fellowship (SFRH/BD/47031/2008) from Fundação para a Ciência e Tecnologia, Portugal. G.A.C. is supported as a fellow by The University of Texas MD Anderson Cancer Center Research Trust and The University of Texas System Regents Research Scholar. Work in Dr Calin’s laboratory is supported in part by grants from the National Institutes of Health (CA135444), the US Department of Defense, and the Pancreatic Cancer Action Network (2009 Seena Magowitz AACR Pilot Grant). STR DNA fingerprinting was done by the Cancer Center Support grant funded Characterized Cell Line core, NCI # CA16672.