Bolsa de Doutoramento FCT: Exploring one-hit-multiple-target strategies for Lung Cancer [2021.07695.BD]

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Exploring One-Hit Multiple Target Strategies for Lung Cancer

Publication . Alexandre, Daniela de Jesus; Cruz, Carla Patrícia Freire Madeira Alves da; Baptista, Pedro Miguel Ribeiro Viana

Non-small cell lung cancer (NSCLC) requires biomarkers that are both sensitive and actionable, as well as network-level therapies. In this context, microRNAs (miRs) have emerged as a unique bridge to meet these needs: their dysregulated signatures discriminate healthy individuals from NSCLC patients, while serving as regulators, each miR can coordinate multiple gene targets, making them compelling therapeutic levers. Leveraging this duality, the present thesis harnesses that property to test a “one-hit, multiple-target” strategy centered on miRs (miR-21, miR-155-3p, miR-3196), integrating sensitive detection with therapeutic modulation. To this end, multiple miRs were initially profiled in NSCLC liquid biopsies (LBs). Subsequently, molecular beacons (MBs) were engineered and qualified for the direct detection of two NSCLC-associated miRs (miR-21/miR-155) in biological samples, laying the groundwork for a diagnostic platform; additionally, one probe was integrated into a microchip, enabling a point-of-care diagnostic prototype. Therapeutically, miR-155-3p was silenced with an antisense strategy, and miR-3196 was reactivated by destabilizing a G-quadruplex (G4) within its precursor using a G4 ligand (phenylpyrrolocytosine; PhpC). To enable efficient intracellular delivery, gold nanoparticles (AuNPs) were developed to deliver both oligonucleotides and PhpC ligand, with complete physicochemical and cellular characterization. Overall, the expression profile analysis identified upregulation of miR-21/miR-155 and downregulation of miR-3196, with clinical correlations. The MB assays achieved high specificity and low-nanomolar sensitivity, enabling rapid readouts. Additionally, the AuNPs targeting miR-155-3p produced efficient cellular uptake and robust knockdown, attenuating oncogenic signaling and outperforming lipid transfection. Moreover, to the best of our knowledge, we have identified, for the first time, a G4-forming region within the human precursor of miR-3196 that restricts its maturation. Subsequently, using the G4 ligand, PhpC, unfolded this structure and restored mature miR-3196. In addition, PhpC-functionalized AuNPs preserved activity, enhanced intracellular persistence, and maintained low acute cytotoxicity across the tested ranges. These studies establish a theragnostic framework for NSCLC: MBs enable minimally invasive detection and real-time pharmacodynamic tracking, while miR modulation, via miR-155 inhibition and G4-guided rescue of miR-3196, may yield broad antitumor effects from a single molecular hit. Furthermore, the AuNP delivery overcomes key barriers to nucleic-acid and small-molecule therapeutics. Collectively, the work advances a translatable, miR-centered one-hit/multi-target paradigm and outlines clear next steps toward in vivo validation and clinical integration.

2026-03-27Tese de doutoramento

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Palavras-chave

Natural sciences, Natural sciences/Biological sciences

Entidade financiadora

Fundação para a Ciência e a Tecnologia, I.P.

Programa de financiamento

Bolsa de Doutoramento

Número da atribuição

2021.07695.BD