BackgroundMicroRNAs (miRNAs) are a class of small regulatory RNAs that target sequences in messenger RNAs (mRNAs) to inhibit their protein output. Dissecting the complexities of miRNA function continues to prove challenging as miRNAs are predicted to have thousands of targets, and mRNAs can be targeted by dozens of miRNAs.ResultsTo systematically address biological function of miRNAs, we constructed and validated a lentiviral miRNA expression library containing 660 currently annotated and 422 candidate human miRNA precursors. The miRNAs are expressed from their native genomic backbone, ensuring physiological processing. The arrayed layout of the library renders it ideal for high-throughput screens, but also allows pooled screening and hit picking. We demonstrate its functionality in both short- and long-term assays, and are able to corroborate previously described results of well-studied miRNAs.ConclusionsWith the miRNA expression library we provide a versatile tool for the systematic elucidation of miRNA function.

Progression from colorectal adenoma to carcinoma is strongly associated with an accumulation of genomic alterations, including gain of chromosome 13. This gain affects the whole q arm and is present in 40%–60% of all colorectal cancers (CRCs). Several genes located at this amplicon are known to be overexpressed in carcinomas due to copy number dosage. A subset of these genes, including the mir-17~92 cluster, are functionally involved in CRC development. The present study set out to explore whether apart from mir-17~92, other miRNAs located at the 13q amplicon show a copy number dependent dosage effect that may contribute to 13q-driven colorectal adenoma-to-carcinoma progression. Integration of publically available miRNA expression, target mRNA expression and DNA copy number data from 125 CRCs yielded three miRNAs, miR-15a, -17, and -20a, of which high expression levels were significantly correlated with a 13q gain and which influenced target mRNA expression. These results could be confirmed by qRT-PCR in a series of 100 colon adenomas and carcinomas.Functional analysis of both mature miRNAs encoded by mir-15a, i.e. miR-15a-5p and miR-15a-3p, showed that silencing of miR-15a-3p significantly inhibited viability of CRC cells. Integration of miR-15a expression levels with mRNA expression data of predicted target genes identified mitochondrial uncoupling protein 2 (UCP2) and COP9 signalosome subunit 2 (COPS2) as candidates with significantly decreased expression in CRCs with 13q gain. Upon silencing of miR-15a-3p, mRNA expression of both genes increased in CRC cells, supporting miR-15a-3p mediated regulation of UPC2 and COPS2 expression. In conclusion, significant overexpression of miR-15a-3p due to gain of 13q is functionally relevant in CRC, with UCP2 and COPS2 as candidate target genes. Taken together our findings suggest that miR-15a-3p may contribute to adenoma-to-carcinoma progression.

Background: In colorectal cancer (CRC) miRNA expression profiling studies have reported differences in expression between colorectal carcinomas and controls, microsatellite stable, microsatellite instable and chromosomal instable tumors, and demonstrated prognostic value for some miRNAs. However, the role of miRNAs in CRC pathogenesis has been only partially investigated. CRC results from the gradual accumulation of multiple genetic and epigenetic changes in the colorectal epithelial cells. The majority of CRCs (85%) shows an accumulation of chromosomal abnormalities. In CRC, up to 15% of the annotated miRNAs are located at regions of chromosomal instability implicated in colorectal adenoma to carcinoma progression. Two of the most frequent chromosomal aberrations in CRC are gain of 13q (>50%) and gain of 20q (>65%). A total of 36 miRNAs are located on these chromosomal arms. However, the leading cause of the miRNA's aberrant expression and the pathways through which they contribute to CRC have not been elucidated. Aim: To investigate the expression of miRNAs located on 13q and 20q in normal colon mucosa, adenomas and CRC. Materials & Methods: DNA and total RNA was isolated from 48 colorectal adenomas, 51 CRCs and 10 normal mucosa samples. Multiplex Ligation-dependent Probe Amplification (MLPA) was used to determine DNA copy number gain of 13q and/or 20q. Expression levels of 17 miRNAs located on 13q and 19 miRNAs on 20q (miRBase release 15) were measured by TaqMan miRNA assays. Results: 11 Of the miRNAs located on 13q and 8 of the miRNAs located on 20q were detected in colon tissue. 15 Of these miRNAs were significantly differentially expressed between colorectal tumors compared to controls and 13 were differentially expressed between carcinomas and adenomas (p<0.05). In addition, 11 miRNAs were significantly differentially expressed between tumors with or without a 13q and/or 20q gain (p<0.05). Conclusion: Gene dosage effects of miRNAs located on chromosomes 13q and 20q play an important role in CRC progression, and taking this into account is essential to achieve a comprehensive understanding of the pathogenesis of this malignancy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4936. doi:10.1158/1538-7445.AM2011-4936