Investigação sobre calicreína plasmática humana em linhagens de tumor de mama: envolvimento com a ativação do plasminogênio e sindecans
Data
2022-11-28
Autores
Shimon, Samara Miyuki Mamede 
Orientadores
Motta, Guacyara da
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Objetivo: Investigar a presença das proteínas do SAP, a PK e os PGHS em linhagens de tumor de mama receptoras de hormônio positivas (MCF-7). Métodos: A expressão de mRNA foi obtida por PCR em tempo real (qPCR) usando primers para as isoformas humanas de PK/PKa, uPA, uPAR e os genes endógenos utilizados foram GADPH e RPL13a. Para analisar a presença de PK, PKa, uPA, uPAR, syn-1 e syn-4 foi utilizada a técnica de microscopia confocal em células não permeabilizadas e permeabilizadas. O ensaio de migração celular na presença de inibidores para PK/PKa e uPA foi feito tanto nas células MCF-7 quanto nas MDA-MB-231 (linhagem triplo negativa). Para analisar a presença das proteínas-alvo (PK/PKa, uPA e uPAR, além do endógeno GAPDH) nas duas linhagens celulares (MCF-7 e MDA-MB-231), foi feita a eletroforese SDS-Page seguida de imunodetecção, utilizando anticorpos específicos. Resultados: A qPCR mostrou que a expressão de uPA (1,28 x 10-3 ± 4,64 x 10-5) foi cerca de 3 vezes mais alta comparada à expressão de seu receptor uPAR (4,65 x 10-4 ± 3,02 x 10-5), a PK/PKa apresentou menor expressão (2,075 x 10-6 ± 1,625 x 10-7) dentre as três. Com a microscopia confocal, foi visto que a uPAR apresenta perfil de receptor de superfície para as duas condições; a uPA é vista em células não permeabilizadas e permeabilizadas, mas mais presente nesta última. A PK/PKa foi identificada no citoplasma da célula, porém não colocalizou com LAMP-1 (marcador lisossomal) e houve identificação da proteína na superfície celular. Syn1 delineou as células na superfície, mas também houve presença de marcação para Syn1 no interior das células. Syn4 mostrou delineamento da superfície das células, mas também apresentou marcação na condição permeabilizada. Os resultados de migração para MCF-7 mostraram que não houve diferença estatística entre o grupo Controle x PKSI (p = 0,6779) e Controle x 4-Cl (p = 0,8814); ou mesmo entre os grupos de inibidores (PKSI x 4-Cl p = 0,5744). Já para a MDA-MB-231, também não houve diferença estatística entre Controle x PKSI (p = 0,8792) ou Controle x 4-Cl (p = 0,6131); mesmo entre os grupos tratados com inibidores também não houve diferença (PKSI x 4-Cl p = 0,5135). Com a eletroforese, foi visto que a uPAR está presente nas células MDA-MB-231, mas não nas MCF-7. A uPA está presente em ambas as linhagens, mas foi visto duas cadeias de uPA em MDA-MB-231. No entanto, PK/PKa é mais presente em células MCF-7. Conclusões: uPA, uPAR e PK/PKa são expressas em MCF-7 e estão localizadas tanto na superfície quanto no citoplasma. A identificação diferenciada de uPAR e PKa em MCF-7 sugere atividade proteolítica que pode influenciar no perfil metastático quando comparado a MDA-MB-231. Os PGHS, Syn1 e Syn4, podem funcionar como receptores putativos para uPA e PK/PKa.
Objective: To investigate the presence of PAS proteins, PK, HSPGs in hormone-positive MCF-7 cell lines. Methods: mRNA expression was evaluated by qPCR using primers to all isoforms of PK/PKa, uPA, uPAR and the endogenous genes, GAPDH and RPL13a. The wound healing assay was made using inhibitors for PK/PKa and uPA on both MCF-7 and MDA-MB-231 cells (triple negative). To analyze the presence of our target proteins (PK/PKa, uPA and uPAR and endogenous GAPDH) in the two cell lines (MCF-7 and MDA-MB-231), SDS-Page electrophoresis with Western blotting was performed using specific antibodies. Results: qPCR showed uPA expression (1.28 x 10-3 ± 4.64 x 10-5) 3-fold higher than the receptor, uPAR (4.65 x 10-4 ± 3.02 x 10-5), PK/PKa showed the lowest expression among the three (2.075 x 10-6 ± 1.625 x 10-7). With confocal microscopy, it was seen that uPAR has a surface receptor profile for both conditions; uPA is observed in non-permeabilized and permeabilized cells, but more present in the permeabilized condition. PK/PKa was identified in the cell cytoplasm, but it did not colocalize with LAMP-1 (lysosomal marker) and the protein was identified on the cell surface. Syn1 outlined the cells on the surface, but there was also the presence of fluorescence for Syn1 inside the cells. Syn 4 showed cell surface delineation, but also fluorescence in the permeabilized condition. The wound healing assay results for MCF-7 showed that there was no statistical difference between the Control x PKSI (p = 0.6779) and Control x 4-Cl (p = 0.8814) groups; or even between the two groups of inhibitors (PKSI x 4-Cl p = 0.5744). As for MDA-MB-231, there was also no statistical difference between Control x PKSI (p = 0.8792) or Control x 4-Cl (p = 0.6131); even between the groups treated with inhibitors there was also no difference (PKSI x 4-Cl p = 0.5135). The electrophoresis showed that uPAR is present in MDA-MB-231 cells, but not in MCF-7. uPA is present in both strains, but two uPA chains were seen in MDA-MB-231. PK/PKa, however, is more present in MCF-7 cells. Conclusions: uPA, uPAR and PK/PKa are expressed in MCF-7 and they are both on the surface and in the cytoplasm. The identification of uPAR and PKa in MCF-7 suggests proteolytic activity that may influence the metastatic profile when compared to MDA-MB-231. The HSPGs, Syn1 and Syn4, can function as putative receptors for uPA and PK/PKa.
Objective: To investigate the presence of PAS proteins, PK, HSPGs in hormone-positive MCF-7 cell lines. Methods: mRNA expression was evaluated by qPCR using primers to all isoforms of PK/PKa, uPA, uPAR and the endogenous genes, GAPDH and RPL13a. The wound healing assay was made using inhibitors for PK/PKa and uPA on both MCF-7 and MDA-MB-231 cells (triple negative). To analyze the presence of our target proteins (PK/PKa, uPA and uPAR and endogenous GAPDH) in the two cell lines (MCF-7 and MDA-MB-231), SDS-Page electrophoresis with Western blotting was performed using specific antibodies. Results: qPCR showed uPA expression (1.28 x 10-3 ± 4.64 x 10-5) 3-fold higher than the receptor, uPAR (4.65 x 10-4 ± 3.02 x 10-5), PK/PKa showed the lowest expression among the three (2.075 x 10-6 ± 1.625 x 10-7). With confocal microscopy, it was seen that uPAR has a surface receptor profile for both conditions; uPA is observed in non-permeabilized and permeabilized cells, but more present in the permeabilized condition. PK/PKa was identified in the cell cytoplasm, but it did not colocalize with LAMP-1 (lysosomal marker) and the protein was identified on the cell surface. Syn1 outlined the cells on the surface, but there was also the presence of fluorescence for Syn1 inside the cells. Syn 4 showed cell surface delineation, but also fluorescence in the permeabilized condition. The wound healing assay results for MCF-7 showed that there was no statistical difference between the Control x PKSI (p = 0.6779) and Control x 4-Cl (p = 0.8814) groups; or even between the two groups of inhibitors (PKSI x 4-Cl p = 0.5744). As for MDA-MB-231, there was also no statistical difference between Control x PKSI (p = 0.8792) or Control x 4-Cl (p = 0.6131); even between the groups treated with inhibitors there was also no difference (PKSI x 4-Cl p = 0.5135). The electrophoresis showed that uPAR is present in MDA-MB-231 cells, but not in MCF-7. uPA is present in both strains, but two uPA chains were seen in MDA-MB-231. PK/PKa, however, is more present in MCF-7 cells. Conclusions: uPA, uPAR and PK/PKa are expressed in MCF-7 and they are both on the surface and in the cytoplasm. The identification of uPAR and PKa in MCF-7 suggests proteolytic activity that may influence the metastatic profile when compared to MDA-MB-231. The HSPGs, Syn1 and Syn4, can function as putative receptors for uPA and PK/PKa.