title: Underexpression of miR-224 in methotrexate resistant human colon cancer cells.
abstract: MicroRNAs (miRNAs) are small non-coding RNAs involved in RNA silencing that play a role in many biological processes. They are involved in the development of many diseases, including cancer. Extensive experimental data show that they play a role in the pathogenesis of cancer as well as the development of drug resistance during treatments. The aim of this work was to detect differentially expressed miRNAs in MTX-resistant cells. Thus, miRNA microarrays of sensitive and MTX-resistant HT29 colon cancer cells were performed. The results were analyzed using the GeneSpring GX11.5 software. Differentially expressed miRNAs in resistant cells were identified and miR-224, which was one of the most differentially expressed miRNAs and with high raw signal values, was selected for further studies. The underexpression of miR-224 was also observed in CaCo-2 and K562 cells resistant to MTX. Putative targets were predicted using TargetScan 5.1 software and integrated with the data from expression microarrays previously performed. This approach allowed us to identify miR-224 targets that were differentially expressed more than 2-fold in resistant cells. Among them CDS2, DCP2, HSPC159, MYST3 and SLC4A4 were validated at the mRNA level by qRT-PCR. Functional assays using an anti-miR against miR-224 desensitized the cells towards MTX, mimicking the resistant phenotype. On the other hand, siRNA treatment against SLC4A4 or incubation of Poly Purine Reverse Hoogsteen (PPRH) hairpins against CDS2 or HSPC159 increased sensitivity to MTX. These results revealed a role for miR-224 and its targets in MTX resistance in HT29 colon cancer cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, E-08028 Barcelona, Spain.
authors.forename: Núria
authors.initials: N
authors.lastname: Mencia
authors.forename: Elisabet
authors.initials: E
authors.lastname: Selga
authors.forename: Véronique
authors.initials: V
authors.lastname: Noé
authors.forename: Carlos J
authors.initials: CJ
authors.lastname: Ciudad
mesh_terms: D020413:3' Untranslated Regions; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms; D019008:Drug Resistance, Neoplasm; D006801:Humans; D020014:K562 Cells; D037102:Lectins / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology*; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; D000964:Antimetabolites, Antineoplastic; C510476:LGALSL protein, human; D037102:Lectins; C546140:MIRN224 microRNA, human; D035683:MicroRNAs; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters; D008727:Methotrexate