Supplementary Data from The Identification of MicroRNAs in a Genomically Unstable Region of Human Chromosome 8q24

Abstract The PVT1 locus is identified as a cluster of T(2;8) and T(8;22) “variant” MYC-activating chromosomal translocation breakpoints extending 400 kb downstream of MYC in a subset (≈20%) of Burkitt's lymphoma (vBL). Recent reports that microRNAs (miRNA) may be associated with fragile sites and cancer-associated genomic regions prompted us to investigate whether the PVT1 region on chromosome 8q24 may contain miRNAs. Computational analysis of the genomic sequence covering the PVT1 locus and experimental verification identified seven miRNAs. One miRNA, hsa-miR-1204, resides within a previously described PVT1 exon (1b) that is often fused to the immunoglobulin light chain constant region in vBLs and is present in high copy number in MYC/PVT1–amplified tumors. Like its human counterpart, mouse mmu-miR-1204 represents the closest miRNA to Myc (∼50 kb) and is found only 1 to 2 kb downstream of a cluster of retroviral integration sites. Another miRNA, mmu-miR-1206, is close to a cluster of variant translocation breakpoints associated with mouse plasmacytoma and exon 1 of mouse Pvt1. Virtually all the miRNA precursor transcripts are expressed at higher levels in late-stage B cells (including plasmacytoma and vBL cell lines) compared with immature B cells, suggesting possible roles in lymphoid development and/or lymphoma. In addition, lentiviral vector-mediated overexpression of the miR-1204 precursor (human and mouse) in a mouse pre–B-cell line increased expression of Myc. High levels of expression of the hsa-miR-1204 precursor is also seen in several epithelial cancer cell lines with MYC/PVT1 coamplification, suggesting a potentially broad role for these miRNAs in tumorigenesis. (Mol Cancer Res 2008;6(2):212–21)

A new species of non-coding RNA, microRNAs (miRNAs) has been identified that may regulate the expression of as many as one third to one half of all protein encoding genes. MicroRNAs are found throughout mammalian genomes, but an association between the location of these miRNAs and regions of genomic instability (or fragile sites) in humans has been suggested [1]. In this review we discuss the possible role of altered miRNA expression on human cancer and conduct an analysis correlating the physical location of murine miRNAs with sites of genetic alteration in mouse models of cancer.

Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study (∼800 siRNAs, ∼400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive.