Gene alteration and precursor and mature microRNA transcription changes contribute to the miRNA signature of primary effusion lymphoma

Abstract

MicroRNAs are regulated by gene alteration, transcription, and processing. Thus far, few studies have simultaneously assessed all 3 levels of regulation. Using real-time quantitative polymerase chain reaction (QPCR)-based arrays, we determined changes in gene copy number, pre-miRNA, and mature miRNA levels for the largest set of primary effusion lymphomas (PELs) to date. We detected PEL-specific miRNA gene amplifications, and concordant changes in pre-miRNA and mature miRNA. We identified 68 PEL-specific miRNAs. This defines the miRNA signature of PEL and shows that transcriptional regulation of pre-miRNA as well as mature miRNA levels contribute nonredundant information that can be used for the classification of human tumors.

Figure 1

Genomic profiling of KSHV and EBV miRNAs. (A) Outline of miRNA maturation and the species that can be detected using pre-miRNA–specific primers and SYBR-based real-time QPCR or miRNA-specific primers and TaqMan-based real-time QPCR. (B) Plot of relative copy number of viral miRNA gene loci. Variations in input DNA were adjusted using U6. This yielded dCT_U6, which represents a logarithmic measure of relative gene copy number. Primers are indicated on the horizontal axis. Positive dCT_U6 indicate lower copy number. Colors indicate different cell lines. The JSC-1 and BC-1 cell lines are EBV positive.

Figure 2

Comparative genomic profiling for cellular miRNA loci in PEL. (A) Variation in miRNA gene copy number. Plotted is the range of dCT_U6 on the vertical and the 5% trimmed mean dCT_U6 on the horizontal axis. The 5% trimmed mean is the arithmetic mean excluding the top and/or bottom 5% of the data. Crosses indicate the values for each cellular miRNA. KSHV miRNAs are shown by open circles; EBV miRNA gene loci, by closed circles; and hsa-miR-34a, by the arrow. Range indicates the difference between the lowest and highest value for each miRNA across all PEL cell lines. If a particular miRNA gene is present in every single cell line at 2 copies per cell, the range is 0 or close to 0, since it reflects only the measurement error. A high range indicates that one or more of the cell lines have more than 2 copies per cell (amplification) or have no copy per cell (deletion). This indicates that one or more cell lines in the sample have sustained amplifications or deletions. These were identified by analyzing the individual scatterplots in Figure S1. (B) Representation of individual clusters of pre-miRNAs as obtained after unsupervised clustering. The axes represent the first 3 principal components of the dataset. The objective of principal component analysis is to test whether all the data are correlated, or if indeed significant patterns or groups of data exist and how many. It serves as a quality control tool for our analysis. As can be seen here, 4, no more and no less, well-separated clusters emerged. These are further analyzed in Figure 3. Individual pre-miRNAs are represented by dots. Cluster membership is indicated by lines and semitransparent hulls.

Figure 3

Pre-miRNA profile of PEL. (A) Unsupervised cluster analysis of standardized dCT_U6 for pre-miRNAs using a correlation metric. Standardization was achieved by calculating Z scores across each individual array of dCT_U6. This technique eliminates variation in assay performance between individual samples. Red indicates higher abundance; blue, lower than median abundance; white, the median. (B,C) Enlarged view. (B) Cluster of pre-miRNAs with decreased abundance in PEL vis-à-vis tonsil. (C) Cluster of pre-miRNAs with increased abundance in PEL vis-à-vis tonsil. (D) Cluster of pre-miRNAs equal abundance in PEL vis-à-vis tonsil. (E) Cluster of pre-miRNAs with high abundance in PEL as well as in nonvirus-associated lymphoma lines. The cluster of absent pre-miRNAs is not shown separately.

Figure 4

Mature miRNA profiles of PEL. (A) Scatterplot analysis of 2 cell lines JSC-1 (vertical axis) and BC-1 (horizontal axis). Plotted are mean CT values of technical triplicates. Lines indicate the SD. Dots correspond to individual miRNAs. CT = 40 indicates absence of a miRNA signal. (B) Variation of mature miRNA levels in PEL. Plotted is the SD across multiple cell lines on the vertical axis against the mean CT on the horizontal axis (n = 10). Open circles indicate mature miRNAs as detected by TaqMan assay. Dark circles indicate that the corresponding pre-miRNA was also overexpressed. Crossed circles indicate miRNAs that were cloned from PEL (data from Cai et al and Samols et al). Shaded area indicates the cutoff used to derive the PEL miRNA signature shown in Table S2. (C) Fraction of total (165) probes on the vertical axis, for which every PEL sample yielded a CT lower than indicated on the horizontal axis. All miRNAs with CT of 38 or less (ie, those that were uniformly detectable in all PEL lines) are tabulated on the right. The # indicates miRNAs that were also cloned once; ##, miRNAs that were cloned independently by 2 groups.^,