Gastroenterology

Gastroenterology

Volume 142, Issue 4, April 2012, Pages 886-896.e9
Gastroenterology

Original Research
Basic and Translational—Alimentary Tract
Strand-Specific miR-28-5p and miR-28-3p Have Distinct Effects in Colorectal Cancer Cells

https://doi.org/10.1053/j.gastro.2011.12.047Get rights and content

Background & Aims

MicroRNAs (miRNAs) can promote or inhibit tumor growth and are therefore being developed as targets for cancer therapies. They are diverse not only in the messenger RNAs (mRNA) they target, but in their production; the same hairpin RNA structure can generate mature products from each strand, termed 5p and 3p, that can bind different mRNAs. We analyzed the expression, functions, and mechanisms of miR-28-5p and miR-28-3p in colorectal cancer (CRC) cells.

Methods

We measured levels of miR-28-5p and miR-28-3p expression in 108 CRC and 49 normal colorectal samples (47 paired) by reverse transcription, quantitative real-time polymerase chain reaction. The roles of miR-28 in CRC development were studied using cultured HCT116, RKO, and SW480 cells and tumor xenograft analyses in immunodeficient mice; their mRNA targets were also investigated.

Results

miR-28-5p and miR-28-3p were down-regulated in CRC samples compared with normal colon samples. Overexpression of miRNAs in CRC cells had different effects and the miRNAs interacted with different mRNAs: miR-28-5p altered expression of CCND1 and HOXB3, whereas miR-28-3p bound NM23-H1. Overexpression of miR-28-5p reduced CRC cell proliferation, migration, and invasion in vitro, whereas miR-28-3p increased CRC cell migration and invasion in vitro. CRC cells overexpressing miR-28 developed tumors more slowly in mice compared with control cells, but miR-28 promoted tumor metastasis in mice.

Conclusion

miR-28-5p and miR-28-3p are transcribed from the same RNA hairpin and are down-regulated in CRC cells. Overexpression of each has different effects on CRC cell proliferation and migration. Such information has a direct application for the design of miRNA gene therapy trials.

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.

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