The colorectal microRNAome

Abstract

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms. The public miRNA database contains 321 human miRNA sequences, 234 of which have been experimentally verified. To explore the possibility that additional miRNAs are present in the human genome, we have developed an experimental approach called miRNA serial analysis of gene expression (miRAGE) and used it to perform the largest experimental analysis of human miRNAs to date. Sequence analysis of 273,966 small RNA tags from human colorectal cells allowed us to identify 200 known mature miRNAs, 133 novel miRNA candidates, and 112 previously uncharacterized miRNA* forms. To aid in the evaluation of candidate miRNAs, we disrupted the Dicer locus in three human colorectal cancer cell lines and examined known and novel miRNAs in these cells. These studies suggest that the human genome contains many more miRNAs than currently identified and provide an approach for the large-scale experimental cloning of novel human miRNAs in human tissues.

Fig. 1.

miRAGE approach for isolation of miRNAs. (A) Schematic of miRAGE method. The approach involves isolation of small RNA species (red ovals), followed by ligation of specialized linkers (white rectangles) that enable robust RT-PCR with biotinylated primers (blue circles). Linkers are enzymatically cleaved and removed by binding to streptavidin-coated magnetic beads (yellow ovals). Released tags are concatenated, cloned, and sequenced. (B) Bioinformatic analyses of miRAGE tags. Tags were grouped together based on a 12-bp internal core sequence. The most highly represented tag in each group was then compared to various RNA databases. Tags not matching known RNA sequences were compared to the human genome and analyzed for precursors with thermodynamically stable hairpin structures (see Materials and Methods for more details).

Fig. 2.

Clustering of miRNAs in the human genome. Analysis of all 133 miRNAs identified 15 that were near other known or novel miRNAs. Yellow boxes represent candidate miRNAs, whereas white boxes represent known miRNAs. Position coordinates are based on National Center for Biotechnology Information Genome Build 35/University of California, Santa Cruz May 2004 assembly.

Fig. 3.

Validation of 133 candidate human miRNAs. A total of 133 miRNA candidates fulfilled expression and biogenesis criteria (black circle). Additional levels of validation include phlyogenetically conserved precursor structures (blue circle), multiple observations of expression (red circle), genomic clustering (yellow circle), observation of corresponding miRNA* forms (green circle), and strong homology to known miRNAs (pink circle).

Fig. 4.

Disruption of human DICER1 helicase domain in colorectal cancer cells. (A) The endogenous locus is shown together with an AAV-Neo targeting construct for insertion into exon 5 of DICER1. HA, homology arm; P, SV40 promoter; Neo, geneticin-resistance gene; R-ITR and L-ITR are right and left inverted terminal repeats; triangles, loxP sites. (B) PCR analysis of parental (+/+), heterozygous (+/Ex5), and homozygous (Ex5/Ex5) clones from DLD1, HCT116, and RKO colorectal cancer cell lines. Primers used for PCR analysis (P1 and P2) are indicated above the endogenous locus in A.

Fig. 5.

miRNA expression in colorectal cancer cells with Dicer disruption. (A) Northern blot analyses show decreased mature miRNAs and increased levels of miRNA precursors in Dicer^ex5 (Ex5) compared with Dicer wild-type (WT) cells using probes for miR-21 and miR-590. (B) Expression levels of known miRNAs as determined by primer-extension quantitative PCR (PE-qPCR), as described (33). For each graph, pairwise comparisons are displayed showing the ratio of expression in Dicer^ex5 to WT clones of each cell type.

Fig. 6.

Discovery of known and novel miRNAs using miRAGE. Each point represents the average number of known or novel miRNAs (y axis) that were identified by analysis of three simulated subsets comprising the number of miRAGE tags indicated (x axis).