Silenciamento do gene itgb3 de células de tumor de mama murino triplo negativo (4T1BM2)
Autor(a) principal: | |
---|---|
Data de Publicação: | 2022 |
Tipo de documento: | Trabalho de conclusão de curso |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFSCAR |
Texto Completo: | https://repositorio.ufscar.br/handle/ufscar/16756 |
Resumo: | Breast cancer is the most recurrent type of tumor in the world, surpassing lung cancer, according to data published in 2021 by the World Health Organization (WHO). When tumor cells spread or invade nearby or distant tissues, secondary tumors are formed and this process is called metastasis. αvβ3 integrin are heterodimeric receptors that connect cells to the extracellular matrix (ECM), they are crucial due to their role in signaling that contributes to cell motility, proliferation and cell behavior, being a therapeutic target of great relevance. The main objective of the present work was the production of a murine breast cancer cell line (4T1BM2) silenced for αvβ3 integrin and the analysis of the effect of this silencing. To obtain this silencing, lentiviral vectors produced from envelope and packaging plasmids were used together with the plasmid encoding the synthesis of interference RNA, which targets part of the β3 subunit gene, to be used in transfection. from the human kidney embryonic cell line (293FT). The viral particles were transduced into the tumor cell and the selection of silenced cells was performed using the antibiotic Puromycin. The verification of partial silencing of β3 integrin was obtained by western blotting and flow cytometry, certifying different levels of integrin in the parental and silenced cells. The evaluation of cellular behavior was carried out through functional migration assays in Boyden's chamber and wound healing. In addition, zymography assays were performed to compare the content of matrix metalloproteinases (MMPs) present in the parental cells and in the silenced cells. The results obtained in this work indicate the achievement of a silencing for the β3 subunit and the functional assays showed a lower migration rate of the silenced cells, as well as a decrease in MMP-9 and an increase in MMP-2 in these cells. These data contribute to improvements in knockdown procedures for this cell type and demonstrate the complex role of integrins in tumor progression. |
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Machado, Aline MariaAraújo, Heloísa Sobreiro Selistre dehttp://lattes.cnpq.br/4065824911933203Altei, Wanessa Fernandahttp://lattes.cnpq.br/3727790337302830http://lattes.cnpq.br/34654065435292534e2aca32-c53d-4d80-950d-5547cb5135f22022-09-30T18:27:53Z2022-09-30T18:27:53Z2022-09-14MACHADO, Aline Maria. Silenciamento do gene itgb3 de células de tumor de mama murino triplo negativo (4T1BM2). 2022. Trabalho de Conclusão de Curso (Graduação em Biotecnologia) – Universidade Federal de São Carlos, São Carlos, 2022. Disponível em: https://repositorio.ufscar.br/handle/ufscar/16756.https://repositorio.ufscar.br/handle/ufscar/16756Breast cancer is the most recurrent type of tumor in the world, surpassing lung cancer, according to data published in 2021 by the World Health Organization (WHO). When tumor cells spread or invade nearby or distant tissues, secondary tumors are formed and this process is called metastasis. αvβ3 integrin are heterodimeric receptors that connect cells to the extracellular matrix (ECM), they are crucial due to their role in signaling that contributes to cell motility, proliferation and cell behavior, being a therapeutic target of great relevance. The main objective of the present work was the production of a murine breast cancer cell line (4T1BM2) silenced for αvβ3 integrin and the analysis of the effect of this silencing. To obtain this silencing, lentiviral vectors produced from envelope and packaging plasmids were used together with the plasmid encoding the synthesis of interference RNA, which targets part of the β3 subunit gene, to be used in transfection. from the human kidney embryonic cell line (293FT). The viral particles were transduced into the tumor cell and the selection of silenced cells was performed using the antibiotic Puromycin. The verification of partial silencing of β3 integrin was obtained by western blotting and flow cytometry, certifying different levels of integrin in the parental and silenced cells. The evaluation of cellular behavior was carried out through functional migration assays in Boyden's chamber and wound healing. In addition, zymography assays were performed to compare the content of matrix metalloproteinases (MMPs) present in the parental cells and in the silenced cells. The results obtained in this work indicate the achievement of a silencing for the β3 subunit and the functional assays showed a lower migration rate of the silenced cells, as well as a decrease in MMP-9 and an increase in MMP-2 in these cells. These data contribute to improvements in knockdown procedures for this cell type and demonstrate the complex role of integrins in tumor progression.O câncer de mama é o tipo de tumor mais recorrente no mundo, superando o de pulmão, segundo dados publicados em 2021 pela Organização Mundial da Saúde (OMS). Quando ocorre o espalhamento ou invasão de células tumorais a tecidos próximos ou distantes são formados tumores secundários e esse processo é denominado metástase. A integrina αvβ3 são receptores heterodiméricos que conectam as células à matriz extracelular (MEC), são cruciais devido ao seu papel nas sinalizações que contribuem para a motilidade celular, proliferação e comportamento celular, sendo um alvo terapêutico de grande relevância. O presente trabalho teve como objetivo principal a produção de uma linhagem celular de câncer de mama murino (4T1BM2) silenciada para a integrina αvβ3 e a análise do efeito desse silenciamento. Para a obtenção deste silenciamento, foram empregados vetores lentivirais produzidos a partir de plasmídeos de envelope e empacotamento em conjunto com o plasmídeo que codifica a síntese de RNA de interferência, o qual tem como alvo parte do gene da subunidade β3, para serem utilizados na transfecção da linhagem celular embrionária de rim humano (293FT). As partículas virais foram transduzidas na célula tumoral e a seleção das células silenciadas foi realizada fazendo uso de antibiótico Puromicina. A verificação do silenciamento parcial da integrina β3 foi obtida por meio de western blotting e citometria de fluxo, certificando diferentes níveis da integrina nas células parentais e silenciadas. Já a avaliação do comportamento celular foi efetivada por meio de ensaios funcionais de migração em câmara de Boyden e wound healing. Além disso, ensaios de zimografia foram realizados para comparar o conteúdo de metaloproteinases de matriz (MMPs) presentes nas células parentais e nas células silenciadas. Os resultados obtidos nesse trabalho indicam o alcance de um silenciamento para a subunidade β3 e os ensaios funcionais demonstraram menor taxa de migração das células silenciadas, bem como a diminuição de MMP-9 e aumento de MMP-2 nessas células. Esses dados contribuem para melhorias nos procedimentos de knockdown para esse tipo celular e demonstram a complexa atuação das integrinas na progressão tumoral.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)porUniversidade Federal de São CarlosCâmpus São CarlosBiotecnologia - BiotecUFSCarAttribution-NonCommercial-NoDerivs 3.0 Brazilhttp://creativecommons.org/licenses/by-nc-nd/3.0/br/info:eu-repo/semantics/openAccessCâncer de mamaMetastasisITGB3Tumor microenvironmentGene silencingBreast cancerIntegrinCIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULARSilenciamento do gene itgb3 de células de tumor de mama murino triplo negativo (4T1BM2)Itgb3 gene silencing in murine triple negative breast tumor cells (4T1BM2)info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/bachelorThesis600600b61211db-d955-45b7-886e-a0cefb89980freponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARORIGINALTCC - Aline Maria Machado.pdfTCC - Aline Maria Machado.pdfTCC Aline Maria Machadoapplication/pdf1161610https://repositorio.ufscar.br/bitstream/ufscar/16756/1/TCC%20-%20Aline%20Maria%20Machado.pdf71a94ffa086871603f97b02372309e9dMD51CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://repositorio.ufscar.br/bitstream/ufscar/16756/2/license_rdfe39d27027a6cc9cb039ad269a5db8e34MD52TEXTTCC - Aline Maria Machado.pdf.txtTCC - Aline Maria Machado.pdf.txtExtracted texttext/plain65837https://repositorio.ufscar.br/bitstream/ufscar/16756/3/TCC%20-%20Aline%20Maria%20Machado.pdf.txt5225ba0f43944e68f3160fd6a36b78ddMD53THUMBNAILTCC - Aline Maria Machado.pdf.jpgTCC - Aline Maria Machado.pdf.jpgIM Thumbnailimage/jpeg6112https://repositorio.ufscar.br/bitstream/ufscar/16756/4/TCC%20-%20Aline%20Maria%20Machado.pdf.jpgcb54bafc6b7c49e5b16afede6e8d88fbMD54ufscar/167562023-09-18 18:32:30.157oai:repositorio.ufscar.br:ufscar/16756Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222023-09-18T18:32:30Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false |
dc.title.por.fl_str_mv |
Silenciamento do gene itgb3 de células de tumor de mama murino triplo negativo (4T1BM2) |
dc.title.alternative.eng.fl_str_mv |
Itgb3 gene silencing in murine triple negative breast tumor cells (4T1BM2) |
title |
Silenciamento do gene itgb3 de células de tumor de mama murino triplo negativo (4T1BM2) |
spellingShingle |
Silenciamento do gene itgb3 de células de tumor de mama murino triplo negativo (4T1BM2) Machado, Aline Maria Câncer de mama Metastasis ITGB3 Tumor microenvironment Gene silencing Breast cancer Integrin CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
title_short |
Silenciamento do gene itgb3 de células de tumor de mama murino triplo negativo (4T1BM2) |
title_full |
Silenciamento do gene itgb3 de células de tumor de mama murino triplo negativo (4T1BM2) |
title_fullStr |
Silenciamento do gene itgb3 de células de tumor de mama murino triplo negativo (4T1BM2) |
title_full_unstemmed |
Silenciamento do gene itgb3 de células de tumor de mama murino triplo negativo (4T1BM2) |
title_sort |
Silenciamento do gene itgb3 de células de tumor de mama murino triplo negativo (4T1BM2) |
author |
Machado, Aline Maria |
author_facet |
Machado, Aline Maria |
author_role |
author |
dc.contributor.authorlattes.por.fl_str_mv |
http://lattes.cnpq.br/3465406543529253 |
dc.contributor.author.fl_str_mv |
Machado, Aline Maria |
dc.contributor.advisor1.fl_str_mv |
Araújo, Heloísa Sobreiro Selistre de |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/4065824911933203 |
dc.contributor.advisor-co1.fl_str_mv |
Altei, Wanessa Fernanda |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://lattes.cnpq.br/3727790337302830 |
dc.contributor.authorID.fl_str_mv |
4e2aca32-c53d-4d80-950d-5547cb5135f2 |
contributor_str_mv |
Araújo, Heloísa Sobreiro Selistre de Altei, Wanessa Fernanda |
dc.subject.por.fl_str_mv |
Câncer de mama Metastasis ITGB3 |
topic |
Câncer de mama Metastasis ITGB3 Tumor microenvironment Gene silencing Breast cancer Integrin CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
dc.subject.eng.fl_str_mv |
Tumor microenvironment Gene silencing Breast cancer Integrin |
dc.subject.cnpq.fl_str_mv |
CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
description |
Breast cancer is the most recurrent type of tumor in the world, surpassing lung cancer, according to data published in 2021 by the World Health Organization (WHO). When tumor cells spread or invade nearby or distant tissues, secondary tumors are formed and this process is called metastasis. αvβ3 integrin are heterodimeric receptors that connect cells to the extracellular matrix (ECM), they are crucial due to their role in signaling that contributes to cell motility, proliferation and cell behavior, being a therapeutic target of great relevance. The main objective of the present work was the production of a murine breast cancer cell line (4T1BM2) silenced for αvβ3 integrin and the analysis of the effect of this silencing. To obtain this silencing, lentiviral vectors produced from envelope and packaging plasmids were used together with the plasmid encoding the synthesis of interference RNA, which targets part of the β3 subunit gene, to be used in transfection. from the human kidney embryonic cell line (293FT). The viral particles were transduced into the tumor cell and the selection of silenced cells was performed using the antibiotic Puromycin. The verification of partial silencing of β3 integrin was obtained by western blotting and flow cytometry, certifying different levels of integrin in the parental and silenced cells. The evaluation of cellular behavior was carried out through functional migration assays in Boyden's chamber and wound healing. In addition, zymography assays were performed to compare the content of matrix metalloproteinases (MMPs) present in the parental cells and in the silenced cells. The results obtained in this work indicate the achievement of a silencing for the β3 subunit and the functional assays showed a lower migration rate of the silenced cells, as well as a decrease in MMP-9 and an increase in MMP-2 in these cells. These data contribute to improvements in knockdown procedures for this cell type and demonstrate the complex role of integrins in tumor progression. |
publishDate |
2022 |
dc.date.accessioned.fl_str_mv |
2022-09-30T18:27:53Z |
dc.date.available.fl_str_mv |
2022-09-30T18:27:53Z |
dc.date.issued.fl_str_mv |
2022-09-14 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/bachelorThesis |
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bachelorThesis |
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publishedVersion |
dc.identifier.citation.fl_str_mv |
MACHADO, Aline Maria. Silenciamento do gene itgb3 de células de tumor de mama murino triplo negativo (4T1BM2). 2022. Trabalho de Conclusão de Curso (Graduação em Biotecnologia) – Universidade Federal de São Carlos, São Carlos, 2022. Disponível em: https://repositorio.ufscar.br/handle/ufscar/16756. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufscar.br/handle/ufscar/16756 |
identifier_str_mv |
MACHADO, Aline Maria. Silenciamento do gene itgb3 de células de tumor de mama murino triplo negativo (4T1BM2). 2022. Trabalho de Conclusão de Curso (Graduação em Biotecnologia) – Universidade Federal de São Carlos, São Carlos, 2022. Disponível em: https://repositorio.ufscar.br/handle/ufscar/16756. |
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https://repositorio.ufscar.br/handle/ufscar/16756 |
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por |
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por |
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600 600 |
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b61211db-d955-45b7-886e-a0cefb89980f |
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Attribution-NonCommercial-NoDerivs 3.0 Brazil http://creativecommons.org/licenses/by-nc-nd/3.0/br/ info:eu-repo/semantics/openAccess |
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Attribution-NonCommercial-NoDerivs 3.0 Brazil http://creativecommons.org/licenses/by-nc-nd/3.0/br/ |
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openAccess |
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Universidade Federal de São Carlos Câmpus São Carlos Biotecnologia - Biotec |
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UFSCar |
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Universidade Federal de São Carlos Câmpus São Carlos Biotecnologia - Biotec |
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