Abstract
Mice lacking the p27Kip1 Cdk inhibitor (Cdkn1b) exhibit increased susceptibility to lymphomas from the Maloney murine leukemia virus (M-MuLV), and exhibit a high frequency of viral integrations at Xpcl1 (Kis2), a locus on the X-chromosome. Xpcl1 encodes miR-106a∼363, a cluster of microRNAs that are expressed in response to adjacent retroviral integrations. We report the first large-scale profile of microRNA expression in MuLV-induced lymphomas, in combination with microarray gene expression analysis. The source material was T-cell lymphomas induced by M-MuLV in p27Kip1 knockout mice and normal thymus. Surprisingly, the overall levels of miRNA expression were equivalent in lymphomas and normal thymus. Nonetheless, the expression of specific microRNAs was altered in tumors. The miR-106a∼363 miRNA were over-expressed in lymphomas, particularly those with viral integrations at the Xpcl1 locus. In contrast, p27Kip1 deletion itself was associated with a different pattern of microRNA expression. Gene expression was dramatically altered in lymphomas, yet paralleled data from T-cell lymphomas induced by other mechanisms. Genes with altered expression in association with the p27Kip1 null genotype were of similar functional classes to those associated with Xpcl1 integration, but with the opposite pattern of expression. Thus, the effect of p27Kip1 deletion may be to oppose an anti-oncogenic effect of Xpcl1 rather than enhancing its oncogenic functions. A subset of miR-106a∼363 target genes was consistently reduced in lymphomas with Xpcl1 integrations, particularly genes with cell cycle and immune functions. We identify four predicted target genes of miR-106a∼363 miRNA, including N-Myc (Mycn), and the TGF-beta receptor (Tgfbr2) using 3'UTR reporter assays. Still, bioinformatic miRNA target predictions were poor predictors of altered gene expression in lymphomas with Xpcl1 integration. Confirmation of miR-106a∼363 gene targeting relevant to the tumor phenotype requires in vivo validation, because only a subset of predicted targets are consistently reduced in tumors that overexpress miR-106a∼363.
Highlights
Mice lacking one or both copies of the p27Kip1 Cdk inhibitor are susceptible to developing a variety of tumor types when exposed to mutagenic agents. [1,2] Several oncogenes and tumor suppressor genes have been shown to cooperate with p27Kip1 loss, including PTEN and APC, in the induction of breast, colon and prostate cancers in mouse models. [2,3,4] We previously identified Xpcl1 as a common viral integration site in T-cell lymphomas induced by Moloney murine leukemia virus (M-MuLV) infection
Retroviral integration sites were previously mapped in these samples, and integrations at the Xpcl1 locus were identified in tumors from p27Kip1 null animals, as well as in tumors arising in mice with one or more intact copies of p27Kip1. [5,6] (The complete dataset is given in Table S1) We first compared the global miRNA expression profile of all tumors in comparison with normal murine thymus
In keeping with what has been reported in human cancers, we found that the majority of microRNAs were reduced in lymphomas relative to total RNA input
Summary
Mice lacking one or both copies of the p27Kip Cdk inhibitor are susceptible to developing a variety of tumor types when exposed to mutagenic agents. [1,2] Several oncogenes and tumor suppressor genes have been shown to cooperate with p27Kip loss, including PTEN and APC, in the induction of breast, colon and prostate cancers in mouse models. [2,3,4] We previously identified Xpcl as a common viral integration site in T-cell lymphomas induced by Moloney murine leukemia virus (M-MuLV) infection. M-MuLV integrations targeted Xpcl at a higher frequently in tumors induced in p27Kip knockout mice. This finding suggested that p27Kip deletion cooperates with Xpcl activation in tumorigenesis, the region was referred to as the X-chromosome p27 cooperating locus. Xpcl is not exclusively targeted by M-MuLV in mice with p27Kip deletions; it has been identified as a viral integration site in p27+/2 and wildtype animals. [9] the normal gene, referred to as Xpcl, apparently produces a primary transcript that is cleaved to form component pre-miRNA hairpins, as well as the flanking ESTs. the individual microRNA comprising miR-106a,363 have been shown to be increased in tumors with viral integrations at the locus. The individual microRNA comprising miR-106a,363 have been shown to be increased in tumors with viral integrations at the locus. [8,10] Large scale viral mutagenesis screens identified increased cointegration of retroviruses at Xpcl and cyclin D3 loci, which further suggests an interaction between the Rb pathway with Xpcl in tumorigenesis. [11]
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