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- Targeting tumour-related alternative splice variant RAC1B with anti-sense oligonucleotidesPublication . Bessa, Cláudia; Gonçalves, Vânia; Jordan, PeterOver 90% of human protein coding genes are able to generate more than one transcript due to alternative splicing. In cancer, certain alternative splicing variants are frequently overexpressed and contribute to tumour progression and aggressiveness. This is well illustrated by RAC1B, a variant of the small GTPase RAC1 that is overexpressed in tumours from colon, breast, lung, pancreas and thyroid. RAC1B is generated by inclusion of alternative exon 3b and the resulting protein contains 19 additional amino acids, which alter regulation and signalling properties. In colorectal cancer, RAC1B is overexpressed in the subgroup of BRAF mutated tumours. Using HT29 cells as a model, RAC1B was shown to exist predominantly in the active GTP-bound conformation in cells and signal via NF-κB to sustain tumour cell survival. To exploit the suitability of this highly gene-specific event for oligonucleotide-based therapeutic intervention, we tested different anti-sense oligonucleotides to interfere with RAC1B expression levels in cancer cells. We found that synthetic siRNAs that induced transcript degradation were able to efficiently suppress RAC1B transcript and protein levels in cells. Phosphorodiamidate morpholino-modifed oligonucleotides (PMO, morpholinos) present higher affinity and stability than conventional oligonucleotides. Thus, a morpholino was designed that targets the intron3–exon3b splice junction including the exonic splice enhancer sequence for SRSF1 binding that we previously identified. When validated in HT29 colorectal tumour cells, this morpholino performed poorly on levels of RAC1B transcript or protein and did not affect RAC1B-dependent cell viability. Indeed, morpholinos do not easily enter mammalian cells in culture but have shown efficacy in animal models in vivo and in human clinical trials. In order to validate, whether the morpholino’s target sequence was able to mediate RAC1B inhibition, we employed the nuclease resistant 2′-O-methyl-modified (2OM) oligonucleotides directed to this and three other exon 3b sequences. We found that all four 2OM oligos reduced RAC1B transcript and protein, comparable to the efficiency of the synthetic siRNA and sustained the downregulating effect for up to 72 h post-transfection.
- Cellular and molecular mechanisms of toxicity of ingested nanomaterialsPublication . Gramacho, Ana Catarina; Rolo, Dora; Martins, Carla; Assunção, Ricardo; Gonçalves, Lídia M.; Bettencourt, Ana; Alvito, Paula; Pereira, Joana; Jordan, Peter; Silva, Maria João; Louro, HenriquetaThe technology based on manufactured nanomaterials (NMs) has been pointed as key enabling technology, due to its potential to improve many products and processes, namely in agriculture, food and feed industry. Many of such products, already available, have NMs such as titanium dioxide nanomaterials (TiO2) and the oral exposure may occur either directly, through the consumption of products/pharmaceuticals containing NMs, or indirectly, through the ingestion of foods contaminated with NMs released from food-contact materials or even through concentration in the food chain due to environmental accumulation. Therefore, the gastrointestinal tract (GIT) appears to be a probable route of exposure to NMs and may lead to systemic exposure if the body barriers are surpassed. One major concern for public health is that NMs may produce adverse outcomes (AO) such as genotoxic effects that are associated with increased risk of cancer. Although NMs have been extensively investigated in recent years, the studies have generated contradictory results, possibly due to differences in the physicochemical properties of the NMs studied and to other variables in the test systems. INSA has previously shown that NMs with the same chemistry, but differing in primary properties may yield different biological effects. Conversely, the NMs properties are context-dependent, i.e. can be affected by the surrounding matrix. These secondary features may be potentially more relevant for determining toxicological outcomes. In particular, processes like digestion may modify the NMs characteristics leading to unexpected toxicity in intestine cells. INGESTnano project aims to investigate the nano-bio interactions of NMs, at cellular and molecular level, in the context of intestinal tract and digestion processes, to better understand their potential negative impacts on human health with special reference to organ-specific cells. TiO2 has been selected as case-study to setup a workflow for addressing nanosafety concerns that may be in the future applied to other NMs to which GIT may be exposed. It is expected that this project will contribute to the safety evaluation of the TiO2 ingested, by elucidating key events (KE) elicited by these NMs and linking exposure to AO.
- Cancer, signaling and alternative splicingPublication . Gonçalves, Vânia; Matos, Paulo; Jordan, PeterAlternative splicing of precursor mRNA is an essential mechanism to increase the complexity of gene expression, and it plays an important role in cellular differentiation and organism development.