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. 2010 Sep;38(17):5919-28.
doi: 10.1093/nar/gkq342. Epub 2010 May 13.

Deep Sequencing Reveals Differential Expression of microRNAs in Favorable Versus Unfavorable Neuroblastoma

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Free PMC article

Deep Sequencing Reveals Differential Expression of microRNAs in Favorable Versus Unfavorable Neuroblastoma

Johannes H Schulte et al. Nucleic Acids Res. .
Free PMC article

Abstract

Small non-coding RNAs, in particular microRNAs(miRNAs), regulate fine-tuning of gene expression and can act as oncogenes or tumor suppressor genes. Differential miRNA expression has been reported to be of functional relevance for tumor biology. Using next-generation sequencing, the unbiased and absolute quantification of the small RNA transcriptome is now feasible. Neuroblastoma(NB) is an embryonal tumor with highly variable clinical course. We analyzed the small RNA transcriptomes of five favorable and five unfavorable NBs using SOLiD next-generation sequencing, generating a total of >188 000 000 reads. MiRNA expression profiles obtained by deep sequencing correlated well with real-time PCR data. Cluster analysis differentiated between favorable and unfavorable NBs, and the miRNA transcriptomes of these two groups were significantly different. Oncogenic miRNAs of the miR17-92 cluster and the miR-181 family were overexpressed in unfavorable NBs. In contrast, the putative tumor suppressive microRNAs, miR-542-5p and miR-628, were expressed in favorable NBs and virtually absent in unfavorable NBs. In-depth sequence analysis revealed extensive post-transcriptional miRNA editing. Of 13 identified novel miRNAs, three were further analyzed, and expression could be confirmed in a cohort of 70 NBs.

Figures

Figure 1.
Figure 1.
NGS of the small RNA transcriptome. (A) Read length distribution (nt) after adapter removal. The y-axis depicts the percentage of read lengths relative to the total number of reads in each dataset, averaged over all datasets. The peak at length 35 is off scale, its actual height is 57%. Read lengths too short for mapping are shown in gray. (B) Proportion of non-coding (green) versus coding (yellow) RNA for each sample. The proportion of ncRNA is >80% in all but two samples analyzed. (C) Distribution of ncRNA species in the samples analyzed. (D) Histogram of Pearson’s correlation coefficients between logarithmic normalized miRNA expression values derived from sequencing versus negative normalized RT-qCt values (15) (displayed for 204 miRNAs available for comparison).
Figure 2.
Figure 2.
Differential miRNA expression between favorable and unfavorable NB is presented as strip chart of normalized read counts. (A,B) Values for absent miRNAs were set to 0.5 to be visible on the logarithmic axis. MiRNA designations as well as raw P-values of t-tests on class means for each miRNA using the logarithmic normalized counts are shown. Additionally, FDR-adjusted P-values are shown. Those were calculated to correct for the number of hypothesis tested, i.e. 10 miRNAs in A and all 465 expressed miRNAs in B. Blue crosses depict EFS datasets and red circles refer to DoD datasets. (A) Expression data (normalized counts) for known NB-associated miRNAs. (B) Expression data (normalized counts) for the 40 best class-separating miRNAs. Rows are sorted according to raw p-values. These data indicate that previously NB-associated miRNAs (e.g. miR-17 and miR542-5p) as well as newly identified miRNAs are differentially expressed between favorable and unfavorable NB. (C) Heat map and cluster dendrogram of the most significant 76 miRNAs (uncorrected P-value <0.05). The EFS (552–556) and DoD (557–561) classes are clearly separated. Clustering was based on Canberra distance and single-linkage clustering. Blue: low expression, yellow: high expression. (D) Histogram of uncorrected p-values after testing equality of expression count means between EFS versus DoD classes for each miRNA. When testing data with equal means, the distribution of P-values is expected to be uniformly distributed across the unit interval (blue line). Here, P-values <0.05 (red line) are enriched.
Figure 3.
Figure 3.
Analysis of expressions of miR/miR* pairs, miR-5p/miR-3p pairs and isomiRs. (A, B, C) Scatter plots are shown, in which each data point (cross, circle, digit) represents expression of a miRNA pair (x-axis: standard or -5p form; y-axis: star or -3p form) in a patient class (blue: EFS, red: DoD). Values for each miRNA pair are presented as log 10 of the geometric average of the five individual patient expression values. The black line indicates the main diagonal x = y. Dotted lines indicate ratios 1:0.15 and 0.15:1. (A) Expression correlation between miRNA-5p and miRNA-3p forms (labeled as 3–9) and standard and star forms (labeled as 1 and 2) for miRNAs known to be involved in NB. (B) Expression correlation between standard and star forms of all expressed miRNAs. (C) Expression correlation between miRNA-5p and miRNA-3p forms of all expressed miRNAs. (D) Global 3′-editing in mature miRNAs. The x-axis indicates the position in each miRNA (formula image corresponds to the most 3′ position). The data are aggregated over all tumors and all miRNAs analyzed and provide a global picture of 3′-editing. Position formula image has the highest overall chance of being different from its reference. The blue curve shows the estimated position-specific sequencing error probability (Supplementary Figure S1). Each blue cross represents one position in SOLiD color space. The sequencing error probability in nucleotide space is considerably lower, as many errors are corrected during conversion from color to nucleotide space.
Figure 4.
Figure 4.
(A) Expression levels of putative new miRNA in favorable (blue) and unfavorable (red) NB, as measured by sequencing. (B) RNA secondary structure of RT-qPCR-validated novel miRNAs as predicted by RNAfold. (C) Kaplan–Meier survival curves for patients with (green) and without (red) Seq 6 expression and low (green) and high (red) Seq 12 expression. For the latter analysis, the 40th percentile was choosen as a cutoff.

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