LUIZ FELIPE TEIXEIRA DA SILVA
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Artigo IPEN-doc 28769 Identification of appropriate housekeeping genes for gene expression studies in human renal cell carcinoma under hypoxic conditions2022 - 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.Artigo IPEN-doc 26882 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 carcinoma2020 - 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.