LUIZ FELIPE TEIXEIRA DA SILVA

LUIZ FELIPE TEIXEIRA DA SILVA

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NF-ĸΒ1 knockout reduces IL6 expression under hypoxia in renal cell carcinoma
2023 - TEIXEIRA, LUIZ F.S.; BELLINI, MARIA H.
Renal cell carcinoma (RCC) is the most common adult renal epithelial cancer, accounting for more than 90% of all renal neoplasms. Clear cell RCC (ccRCC) is the most common subtype of RCC. Most patients with ccRCC have a mutation in the von Hippel-Lindau (VHL) tumor suppressor gene, which encodes a protein that downregulates various intracellular proteins, including hypoxia-inducible factor (HIF). Many molecules have been identified to be responsible for the aggressive phenotype of ccRCC, including the transcription factor nuclear factor kappa B (NF-кB). The increase in NF-кB activity observed in RCC is correlated with an increase in angiogenesis markers, such as interleukin 6 (IL-6). In recent years, several groups have demonstrated the functional role of NF-кB1 in RCC tumorigenicity. Herein, we used the CRISPR/Cas-9 technique to obtain an NF-кB1 knockout-human renal adenocarcinoma cell line. Expression of IL-6 at the mRNA and protein levels was analyzed under normoxia and hypoxia by real time-polymerase chain reaction and multiplex assay, respectively. The CRISPR/Cas9 technique was effective in producing 786-0 knockout cells for NF-κB1 (p105/p50), as confirmed by western blot analysis. Suppression of p50 expression in 786-0 single guide RNA (sg)1, 786-0 sg2 and 786-0 sg3 cells downregulated IL-6 mRNA and protein expression under normoxia and hypoxia. The observed decrease in the differential expression of IL-6 in hypoxia/normoxia is suggestive of a change in cellular responsiveness to hypoxia with respect to IL-6.
Identification of appropriate housekeeping genes for gene expression studies in human renal cell carcinoma under hypoxic conditions
2022 - TEIXEIRA, LUIZ F.S.; GIGLIOTTI, RODRIGO; FERREIRA, LUANA da S.; BELLINI, MARIA H.
Background: Hypoxia pathways are deregulated in clear renal cell carcinoma (ccRCC) because of the loss of the von Hippel-Lindau tumor suppressor function. Quantitative PCR is a powerful tool for quantifying differential expression between normal and cancer cells. Reliable gene expression analysis requires the use of genes encoding housekeeping genes. Therefore, in this study, eight reference candidate genes were evaluated to determine their stability in 786-0 cells under normoxic and hypoxic conditions. Methods and Results: Four different tools were used to rank the most stable genes—geNorm, NormFinder, BestKeeper, and Comparative Ct (ΔCt), and a general ranking was performed using RankAggreg. According to the four algorithms, the TFRC reference gene was identified as the most stable. There was no agreement among the results from the algorithms for the 2nd and 3rd positions. A general classification was then established using the RankAggreg tool. Finally, the three most suitable reference genes for use in 786-0 cells under normoxic and hypoxic conditions were TFRC, RPLP0, and SDHA. Conclusions: To the best of our knowledge, this is the first study to identify reliable genes that can be used for gene expression analysis in ccRCC in a hypoxic environment.
Avaliação da capacidade angiogênica da linhagem celular de adenocarcinoma renal humano knockout para a proteína NF-kB1
2021 - SILVA, LUIZ F.T. da
O carcinoma de células renais (CCR) é o câncer epitelial renal adulto mais comum, sendo responsável por mais de 90% de todas as neoplasias renais. O subtipo mais frequente de CCR é o de células claras (CCRcc). A maioria dos pacientes com CCRcc possui mutação no gene supressor tumoral de Von Hippel-Lindau (VHL). O gene VHL codifica uma proteína, a VHL, que é capaz de regular negativamente uma série de proteínas intracelulares, dentre elas o fator induzível por hipóxia (HIF). Muitas moléculas têm sido apontadas como responsáveis pelo fenótipo agressivo desse tumor, uma delas é o fator de transcrição NF-kB, que é o nome coletivo para os fatores de transcrição da família Rel. Em mamíferos são conhecidos cinco membros desta família: RelA (p65), RelB, c-Rel, NF-kB1 (p105/p50) e o NF-kB2 (p100/p52). No CCR, o aumento da atividade do NF-kB correlacionava-se com o aumento de marcadores de angiogênese tais como o fator de crescimento endotelial vascular (VEGF) e Interleucina 6 (IL-6). Nos últimos anos vários grupos têm demonstrado o papel funcional do NF-kB1 na tumorigenicidade do CCR. Nesse projeto utilizamos a técnica CRISPR/Cas-9 para obtenção de uma linhagem celular de adenocarcinoma renal humano knockout para a proteína NF-kB1. Foi realizada a verificação do gene endógeno mais estável para condições de normóxia e hipóxia. Foi feita a quantificação dos níveis de VEGF e IL-6 em condição de normóxia e hipóxia. A técnica CRISPR/Cas9 foi eficaz para obtenção de células 786-0 nocaute para NF-kB1 (p105/p50). Dentre os oito genes endógenos analisados, o TRFC foi o mais estável e, consequentemente, o mais adequado para estudos com as células 786-0 em hipóxia. A supressão da expressão da p50 nos clones 786-0 sg1, 786-0 sg2 e 786-0 sg3 resultou na redução de VEGF e IL6 tanto em normóxia quando em hipóxia. A redução do diferencial hipóxia/normóxia demonstra uma alteração na responsividade celular à hipóxia no que diz respeito à Il-6. A redução dos níveis de IL-6, pode justificar a redução da MMP-9 e migração celular observados por nosso grupo.
Silencing of nuclear factor kappa b 1 gene expression inhibits colony formation, cell migration and invasion via the downregulation of interleukin 1 beta and matrix metallopeptidase 9 in renal cell carcinoma
2020 - TEIXEIRA, LUIZ F.S.; PERON, JEAN P.S.; BELLINI, MARIA H.
Renal cell carcinoma (RCC) is a highly deadly urological tumor due to its high metastatic incidence and its notorious chemoresistance. The nuclear transcription factor kappa B (NF-κB) family has been associated with apoptosis resistance and cellular invasion in RCC. The purpose of this study was to evaluate the impact of NF-κB1 gene silencing on the colony formation, cell migration and invasion abilities of the RCC cell line. Renca–mock and Renca-shRNA-NF-κB1 cells were used in this work. NF-κB1 downregulation was assessed by western blotting. The mRNA expression levels of interleukin-1 beta (IL-1β) and MMP-9 were assessed by real-time quantitative polymerase chain reaction (RT-qPCR). The IL-1β levels in the culture media were determined by a commercial ELISA kit. The MMP-9 protein expression and gelatinolytic activity were evaluated by western blotting and zymography, respectively, and the migration and invasion abilities were analysed. The expression levels of p105 and p50 in Renca-shRNA-NF-κBmoc1 cells were significantly reduced compared with those in the Renca–mock cells. The colony numbers of shRNA-NF-кB1 cells were lower than the colony numbers of the Renca– mock cells. NF-κB1 knockdown inhibited the cell migration and invasion of Renca-shRNA-NF-κB1 cells. These cells also exhibited reduced levels of IL-1β. The MMP-9 expression and activity levels were significantly reduced in Renca-shRNANF- κB1 cells. Taken together, these results indicate that the downregulation of NF-κB1 suppresses the tumourigenicity of RCC by reducing MMP-9 expression and activity; thus, NF-κB1 could be a molecular target for RCC treatment.
Knockdown of NF-κB1 by shRNA inhibits the growth of renal cell carcinoma in vitro and in vivo
2018 - IKEGAMI, AMANDA; TEIXEIRA, LUIZ F.S.; BRAGA, MARINA S.; DIAS, MATHEUS H. dos S.; LOPES, EDUARDO C.; BELLINI, MARIA H.
Renal cell carcinoma (RCC) accounts for approximately 2-3% of human malignancies and is the most aggressive among urologic tumors. Biological heterogeneity, drug resistance and chemotherapy side effects are the biggest obstacles to the effective treatment of RCC. The NF-кB transcription factor is one of several molecules identified to be responsible for the aggressive phenotype of this tumor. In the past decade, several studies have demonstrated the activation of NF-kB in RCC, and many implicated NF- κB1 (p50) as an important molecule in tumor progression and metastasis. In the present study, a lentivirus was used to deliver shRNA targeting NF-κB1 into mouse renal cell carcinoma (Renca) cells. It was determined that the knockdown of the NF-κB1 gene led to a reduction in cell proliferation and late apoptosis/necrosis in vitro. Flow cytometry analysis demonstrated G2/M arrest in the cells. In addition, immunoblotting analysis revealed a significant increase in cyclin B1 and Bax. In vivo experiments showed that Renca-shRNA-NF-кB1 cells have significantly diminished tumorigenicity. Moreover, immunohistochemical analysis revealed an increase in necrotic areas of Renca-shRNANF- кB1 tumors. Thus, this study indicates that downregulation of NF-кB1 can suppress RCC tumorigenesis by inducing late apoptosis/necrosis. Therefore, NF-кB1 may be a potential therapeutic target for RCC.
Avaliação da capacidade clonogênica, migratória e invasiva de linhagem celular de carcinoma renal após o silenciamento do gene NF-κβ1
2016 - SILVA, LUIZ F.T. da; BELLINI, MARIA H.