The cDNA microarray is one technological approach that has the potential to accurately measure changes in global mRNA expression levels. We report an assessment of an optimized cDNA microarray platform to generate accurate, precise and reliable data consistent with the objective of using microarrays as an acquisition platform to populate gene expression databases. The study design consisted of two independent evaluations with 70 arrays from two different manufactured lots and used three human tissue sources as samples: placenta, brain and heart. Overall signal response was linear over three orders of magnitude and the sensitivity for any element was estimated to be 2 pg mRNA. The calculated coefficient of variation for differential expression for all non-differentiated elements was 12-14% across the entire signal range and did not vary with array batch or tissue source. The minimum detectable fold change for differential expression was 1.4. Accuracy, in terms of bias (observed minus expected differential expression ratio), was less than 1 part in 10 000 for all non-differentiated elements. The results presented in this report demonstrate the reproducible performance of the cDNA microarray technology platform and the methods provide a useful framework for evaluating other technologies that monitor changes in global mRNA expression.
Judging from the large number of integrations into the Pvt1 locus - more than in the nearby Myc locus - Pvt1 and the microRNAs encoded by it are as important as Myc in T lymphomagenesis, and, presumably, in T cell activation. An analysis of the co-mutations in the lymphomas likely place Pvt1 and Myc into the same pathway.
BackgroundGPR110 is an orphan G protein-coupled receptor--a receptor without a known ligand, a known signaling pathway, or a known function. Despite the lack of information, one can assume that orphan receptors have important biological roles. In a retroviral insertion mutagenesis screen in the mouse, we identified GPR110 as an oncogene. This prompted us to study the potential isoforms that can be gleaned from known GPR110 transcripts, and the expression of these isoforms in normal and transformed human tissues.MethodsVarious epitope-tagged isoforms of GPR110 were expressed in cell lines and assayed by western blotting to determine cleavage, surface localization, and secretion patterns. GPR110 transcript and protein levels were measured in lung and prostate cancer cell lines and clinical samples, respectively, by quantitative PCR and immunohistochemistry.ResultsWe found four potential splice variants of GPR110. Of these variants, we confirmed three as being expressed as proteins on the cell surface. Isoform 1 is the canonical form, with a molecular mass of about 100 kD. Isoforms 2 and 3 are truncated products of isoform 1, and are 25 and 23 kD, respectively. These truncated isoforms lack the seven-span transmembrane domain characteristic of GPR proteins and thus are not likely to be membrane anchored; indeed, isoform 2 can be secreted. Compared with the median gene expression of ~200 selected genes, GPR110 expression was low in most tissues. However, it had higher than average gene expression in normal kidney tissue and in prostate tissues originating from older donors. Although identified as an oncogene in murine T lymphomas, GPR110 is greatly overexpressed in human lung and prostate cancers. As detected by immunohistochemistry, GPR110 was overexpressed in 20 of 27 (74%) lung adenocarcinoma tissue cores and in 17 of 29 (59%) prostate adenocarcinoma tissue cores. Additionally, staining with a GPR110 antibody enabled us to differentiate between benign prostate hyperplasia and potential incipient malignancy.ConclusionOur work suggests a role for GPR110 in tumor physiology and supports it as a potential therapeutic candidate and disease marker for both lung and prostate cancer.
Retroviruses can cause tumors when they integrate near a protooncogene or tumor suppressor gene of the host. We infected >2,500 mice with the SL3-3 murine leukemia virus; in 22 resulting tumors, we found provirus integrations nearby or within the gene that contains the mir-17-92 microRNA (miRNA) cistron. Using quantitative real-time PCR, we showed that expression of miRNA was increased in these tumors, indicating that retroviral infection can induce expression of oncogenic miRNAs. Our results demonstrate that retroviral mutagenesis can be a potent tool for miRNA discovery.oncogene ͉ retroviral mutagenesis M icroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression. They are initially transcribed by RNA polymerase II and contained within hairpins on a long primary transcript. The hairpins are then processed by two successive steps mediated by a double-stranded RNA-binding protein and RNase III (in mammals, DGCR8 and Drosha followed by TRBP and Dicer), to create the mature Ϸ21-nt miRNA. The miRNA is loaded into the RNA-induced silencing complex and in animals, the complex is directed to mRNAs by the complementarity of six or seven bases within the miRNA. This leads to either translational repression or mRNA cleavage (1). It has been predicted that one-third of human genes may be regulated in this way (2).Several miRNA hairpins can be encoded as a cistron on a single primary transcript. Such is the case for the human gene, c13orf25, and its mouse homolog. Here, a primary transcript encodes (in order, 5Ј to 3Ј) used a miRNA microarray to profile human B cell tumor lines and found that miRNAs encoded by c13orf25 were overexpressed. They then showed that in mice, when B cells constitutively overexpressing c-Myc were transduced with part of the human cistron containing miRNAs 17-3p to 19b-1, lymphoma formed at an accelerated pace, suggesting that these miRNAs could be oncogenes (3). In addition, Hayashita et al. (4) found that the mir-17-92 cistron was overexpressed in human lung cancer. Also, O'Donnell et al. (5) showed that c-Myc expression leads to increased expression of miRNAs from the mir-17-92 cistron, and that mir-17-5p and mir-20 negatively regulate the cell proliferation factor E2F1, suggesting that these miRNAs could also have tumor suppressor properties.Although microarray analysis of miRNA expression in tumors has proven quite useful in identifying candidates involved in cancer and has provided seminal insight, this method inherently cannot distinguish cause from correlation and so must be corroborated by additional data, expression of a transgene or identification of implicating deletions, translocations, and other mutations (3, 6-8). Alternatively, retroviral insertional mutagenesis might be used to identify causative cancer genes. In this method, slow-transforming retroviruses, which themselves carry no oncogene, insert provirus DNA into the host DNA. Because the provirus integrates into essentially random locations in the host genome, retroviruses can be used as a gene discovery tool ...
Retroviral insertion into a host genome is a powerful tool not only for the discovery of cancer genes, but also for the discovery of potential oncogenic noncoding RNAs. In a large-scale mouse T lymphocyte tumor screen we found a high density of integrations upstream of the mir-106a microRNA cistron. In tumors containing an integration, the primary transcript encoding the mir106a cistron was overexpressed five to 20-fold compared with that of control tumors; concomitantly, the mature mir-106a and mir-363 microRNAs were highly overexpressed as well. These findings suggest the mir-106a cistron plays an important role in T cell tumorigenesis. FindingsRetroviral insertions into the genome of a host can induce tumor formation by altering gene expression or function. Integration of a retrovirus near a gene can induce overexpression of the gene through the viral promoter or enhancer, while insertion of a retrovirus into a gene can cause both activation and inactivation. If the affected genes are proto-oncogenes or tumor suppressor genes, the insertion events may lead to tumor formation [1]. Consequently, retroviral mutagenesis has been used to search entire genomes for genes involved in cancer development [2][3][4], including oncogenic microRNAs (miRNAs) [5]. MiRNAs are short (~22 bp) noncoding RNAs that are implicated in gene regulation and cancer [6][7][8][9][10]. In a large-scale retroviral insertion mutagenesis screen, we used the murine leukemia virus (MLV) strain SL3-3, which causes T lymphomas [11], and identified several miRNAs that are potentially involved in tumorigenesis.We previously demonstrated that a group of these retroviral insertions induces overexpression of the oncogenic mmu-mir-17 miRNA cistron in mouse tumors [5]. Here we build on our validation of the retrovirus insertional mutagenesis method to identify oncogenic miRNA and present another potentially oncogenic miRNA cistron, mmu-mir-106a. In this screen, male BALB/c mice were treated with ethyl-nitroso-urea (ENU) and bred to normal female mice. ENU treatment was conducted to increase the recovery of tumor suppressors in the F1 progeny through mutagenesis of the paternal allele. Newborn offspring mice were then injected with MLV strain SL3-3. After becoming moribund due to tumor development, mice were euthanized and thymus and spleen tissues were collected and stored at -80°C. Locations of the SL3-3 provirus integration sites were identified as previously described using a splinkerette based PCR method [3] that amplifies genomic DNA flanking the 5' LTR of the virus.
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