BackgroundRetrotransposons are mobile elements that have a high impact on shaping the mammalian genomes. Since the availability of whole genomes, genomic analyses have provided novel insights into retrotransposon biology. However, many retrotransposon families and their possible genomic impact have not yet been analysed.ResultsHere, we analysed the structural features, the genomic distribution and the evolutionary history of mouse VL30 LTR-retrotransposons. In total, we identified 372 VL30 sequences categorized as 86 full-length and 49 truncated copies as well as 237 solo LTRs, with non-random chromosomal distribution. Full-length VL30s were highly conserved elements with intact retroviral replication signals, but with no protein-coding capacity. Analysis of LTRs revealed a high number of common transcription factor binding sites, possibly explaining the known inducible and tissue-specific expression of individual elements. The overwhelming majority of full-length and truncated elements (82/86 and 40/49, respectively) contained one or two specific motifs required for binding of the VL30 RNA to the poly-pyrimidine tract-binding protein-associated splicing factor (PSF). Phylogenetic analysis revealed three VL30 groups with the oldest emerging ~17.5 Myrs ago, while the other two were characterized mostly by new genomic integrations. Most VL30 sequences were found integrated either near, adjacent or inside transcription start sites, or into introns or at the 3′ end of genes. In addition, a significant number of VL30s were found near Krueppel-associated box (KRAB) genes functioning as potent transcriptional repressors.ConclusionCollectively, our study provides data on VL30s related to their: (a) number and structural features involved in their transcription that play a role in steroidogenesis and oncogenesis; (b) evolutionary history and potential for retrotransposition; and (c) unique genomic distribution and impact on gene expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13100-016-0066-8) contains supplementary material, which is available to authorized users.
Retrotransposons copy their sequences via an RNA intermediate, followed by reverse transcription into cDNA and random insertion, into a new genomic locus. New retrotransposon copies may lead to cell transformation and/or tumorigenesis through insertional mutagenesis. Methylation is a major defense mechanism against retrotransposon RNA expression and retrotransposition in differentiated cells, whereas stem cells are relatively hypo-methylated. Epithelial-to-mesenchymal transition (EMT), which transforms normal epithelial cells into mesenchymal-like cells, also contributes to tumor progression and tumor metastasis. Cancer stem cells (CSCs), a fraction of undifferentiated tumor-initiating cancer cells, are reciprocally related to EMT. In the present study, the outcome of long terminal repeat (LTR)-Viral-Like 30 (VL30) retrotransposition was examined in mouse mammary stem-like/progenitor HC11 epithelial cells. The transfection of HC11 cells with a VL30 retrotransposon, engineered with an EGFP-based retrotransposition cassette, elicited a higher retrotransposition frequency in comparison to differentiated J3B1A and C127 mouse mammary cells. Fluorescence microscopy and PCR analysis confirmed the specificity of retrotransposition events. The differentiated retrotransposition-positive cells retained their epithelial morphology, while the respective HC11 cells acquired mesenchymal features associated with the loss of E-cadherin, the induction of N-cadherin, and fibronectin and vimentin protein expression, as well as an increased transforming growth factor (TGF)-β1, Slug, Snail-1 and Twist mRNA expression. In addition, they were characterized by cell proliferation in low serum, and the acquisition of CSC-like properties indicated by mammosphere formation under anchorage-independent conditions. Mammospheres exhibited an increased Nanog and Oct4 mRNA expression and a CD44 + /CD24 -/low antigenic phenotype, as well as self-renewal and differentiation capacity, forming mammary acini-like structures. DNA sequencing analysis of retrotransposition-positive HC11 cells revealed retrotransposed VL30 copies integrated at the vicinity of EMT-, cancer type-and breast cancer-related genes. The inoculation of these cells into Balb/c mice produced cytokeratin-positive tumors containing pancytokeratin-positive cells, indicative of cell invasion features. On the whole, the findings of the present study demonstrate, for the first time, to the best of our knowledge, that stem-like epithelial HC11 cells are amenable to VL30 retrotransposition associated with the induction of EMT and CSC generation, leading to tumorigenesis.
Tinzaparin is an anticoagulant and antiangiogenic drug with inhibitory properties against tumor growth. VEGF stimulates angiogenesis, while an association between reactive oxygen species (ROS) and angiogenesis is involved in tumor progression. The present study aimed to investigate the effect of tinzaparin on VL30 retrotransposition-positive mouse HC11 mammary stem-like epithelial cells, previously reported to be associated with induced mammosphere/cancer stem cell (CSC) generation and tumorigenesis. Under 24 h serum starvation, 15.2% nominal retrotransposition frequency was increased to 29%. Additionally, while treatment with 3–12 ng/ml VEGF further induced retrotransposition frequency in a dose-dependent manner (up to 40.3%), pre-incubation with tinzaparin (2 IU/ml) for 0.5–4 h reduced this frequency to 18.3% in a time-dependent manner, confirmed by analogous results in NIH3T3 fibroblasts. Treatment with 10–40 pg/ml glucose oxidase (GO) for 24 h induced HC11 cell retrotransposition in a dose-dependent manner (up to 82.5%), while a 3 h pre-incubation with tinzaparin (1 or 2 IU/ml) elicited a 13.5 or 25.5% reduction in retrotransposition, respectively. Regarding tumorigenic VL30 retrotransposition-positive HC11 cells, treatment with 2 IU/ml tinzaparin for 5 days reduced proliferation rate in a time-dependent manner (up to ~55%), and after 3 weeks, disaggregated soft agar-formed foci, as well as low-adherent mammospheres, producing single mesenchymal-like cells with a ~50% reduced retrotransposition. With respect to the VL30 retrotransposition mechanism: While 12 ng/ml VEGF increased the level of VL30 and endogenous reverse transcriptase (enRT) transcripts ~1.41- and ~1.16-fold, respectively, subsequent tinzaparin treatment reduced both endogenous/ROS- and VEGF-induced levels 1.15- and 0.40-fold (VL30) and 0.60- and 0.52-fold (enRT), respectively. To the best of our knowledge, these data demonstrate for the first time, the novel inhibition activity of tinzaparin against ROS- and VEGF-induced VL30 retrotransposition, and the proliferation and/or aggregation of mouse HC11 mammosphere/tumor-initiating CSCs, thus contributing to the inhibition of VL30 retrotransposition-induced primary tumor growth.
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