Further insights into regucalcin actions as a potential tumour suppressor in human prostate

• Autor(a) principal: Fonseca, Lara Raquel dos Santos
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/11266
• topic: Apoptose; Cancro da Próstata; Metastização; Proliferação; Regucalcina; Sobrevivência; Domínio/Área Científica::Engenharia e Tecnologia::Bioquímica

The calcium-binding protein regucalcin (RGN) regulates several biological processes, namely the hallmarks of cancer, such as cell proliferation and apoptosis. Previous work of our research group reported that the loss of RGN expression accompanies the onset and progression of prostate cancer (PCa). However, it remains largely unknown if the decrease of RGN expression is a cause or consequence of PCa. The present thesis aims to investigate the relationship of RGN expression levels with the hallmarks of cancer and PCa patients’ outcomes. An in silico analysis using the CancerTool3 software and patients’ datasets demonstrated that the loss of RGN correlates with the onset and progression of PCa to metastatic forms of disease. However, no correlation was found between the expression levels of RGN and the histological score of Gleason or PCa recurrence. Moreover, it was found that PCa patients with higher RGN expression levels displayed higher disease-free survival. Bioinformatic analysis of gene-to-gene expression correlation showed that the RGN gene expression in primary prostate tumours correlates directly with the expression of cyclin-dependent kinase inhibitor 1A (CDKN1A) and IL6 genes. CDKN1A gene encodes the cell cycle inhibitor p21, which has been indicated as a target of RGN in rat prostate and human cancers. No other previous study has identified a connection of RGN with IL-6 in cancer cells or tissues. Secondly, we investigated whether the loss of RGN expression may alter human prostate cell fate. Using a siRNA gene silencing approach, RGN gene expression in human non-neoplastic PNT1A cells was knockdown (KD), which was confirmed by quantitative real-time PCR. RGN gene KD increased the viability and proliferative ability of PNT1A cells as indicated by the MTT and sulforhodamine B assays’ results, and the fluorescent immunocytochemistry of the Ki-67 proliferation marker. The reduced caspase-3-like activity found in PNT1A cells KD for RGN demonstrated that the loss of this protein might contribute to apoptosis resistance. Western Blot results showed that the changes in prostate cell fate were accompanied by the altered expression of oncoproteins, such as AKT and its phosphorylated form, as well as c-myc; and also key regulators of the extrinsic pathway of apoptosis, namely, FasR, FasL and caspase-8. RGN gene KD did not alter the glycolytic metabolism of PNT1A cells, as indicated by the results of glucose consumption and lactate production. Altogether, the present findings showed that the loss of RGN expression alters the behaviour of human prostate cells and promotes the aggressiveness and progression of PCa, which supports the RGN’s role as a tumour suppressor protein. Moreover, the obtained results also support the idea that maintaining high RGN expression levels may prevent the prostate carcinogenic process and delay the progression of the disease.