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Supporting materials
The following supplementary materials are available:
Supplementary Figure 1. Bioinformatic assessment of miR-22 as a potential tumor
suppressor gene. (a) Location of miR-22 in a minimal LOH (loss of heterozygosity) region at
17p13.3 between markers D17S1866 and D17S1574 in cancers. (b) Genomic organization of
the human miR-22 locus. (c) RNA editing of miR-22 precursor. Bold bases form mature miR22; boxed sequence is miR-22 seed region; red bases with arrows are prone to editing. (d)
Phylogenetic conservation of the non-coding region containing miR-22. Vista was used to
generate pairwise alignments between the human genomic sequence (March 2006 assembly)
and those from the indicated species. The graph is a plot of nucleotide identity for a 100-bp
sliding window centered at a given position. Annotated transcripts produced from this locus
are shown at the top. Note that the 5’ end of miR-22 is toward the right, and the miR-22containing region is in the red box. Red indicates a conserved non-coding region; blue
indicates a conserved exon; turquoise indicates conserved UTRs. (e) Plot representing
evolutionary conservation taken from the UCSC Genome Browser (human genome March
2006 assembly).
Supplementary Figure 2. Sequences of synthetic miR-22 duplex and negative control RNA
duplex. The sequence for the mature miR-22 is underlined.
Supplementary Figure 3. Relative levels of miR-22 and control RNA measured by
quantitative RT-PCR. (a and e) Relative control RNA levels after transfection and colony
formation. Colonies formed from control RNA-transfected cells indicated as Ctrl-treated
colonies. (b, c, d and f) Relative miR-22 levels after miR-22 or anti-miR-22 transfection. Data
are presented as mean ± SD from at least three independent experiments. *, P < 0.05; **, P <
0.01; ***, P < 0.001.
Supplementary Figure 4. The quantitative RT-PCR products for U6 and miR-22 detection in
an agarose gel. The PCR primers were shown to generate a single amplification band.
Supplementary Table 1. Expression changes obtained by microarray analysis on MCF-7
cells treated with miR-22 compared to control RNA. The experimental procedures for
microarray assay are described below. MCF-7 cells were transfected with 50 nM negative
control RNA or miR-22. Total RNA was isolated using TRI Reagent (Sigma, St. Louis, MO)
24 hours after transfection, and the gene expression profiles were analyzed using an Affymetrix
Human Genome U133 plus 2.0 array. In brief, 1 μg of total RNA was used to synthesize double-
stranded cDNA. Biotin-labeled cRNA produced by in vitro transcription of double-stranded
cDNAs using Custom MessageAmp II-Biotin (Ambion, Austin, TX) were fragmented into
strands 35–200 bases long. A hybridization cocktail including fragmented cRNAs was first
subjected to 99°C for 5 minutes of denaturization and then hybridized to an array for 16 hours
at 45°C. After washing and staining on an Affymetrix Fluidics Station 450, the array was
scanned using GeneChip Scanner 3000. Raw data of gene expression signals were extracted
using Affymetrix GeneChip Operating Software Version 1.4. Normalization and expression
analysis were performed with a DNA-chip analyzer (dChip). Invariant set normalization was
used to normalize arrays at the probe level. The microarray data have been submitted to the
Gene Expression Omnibus public database at the National Center for Biotechnology
Information, following the Minimum Information About a Microarray Gene Experiment
guidelines. The accession number is GSE17508. GO analysis was performed using MAS
(Molecular Annotation System) 2.0 software from CapitalBio (Beijing, China).
Supplementary Table 2. Affymetrix gene expression and bioinformatic analysis of genes
down-regulated more than 1.4-fold in MCF7 cells treated with miR-22 compared to control
RNA. Analysis results of genes down-regulated more than 1.4-fold (a) and the predicted
consequential pairing of miR-22 seed region and the conserved target region within LRRC1/
DPM2/ NPNT/ HNRNPA3/ MYCBP across mammalian species (b) were shown.
Supplementary Table 3. Primers for cloning of MYCBP 3’-UTR and coding region, and for
quantitative RT-PCR.