O microambiente de hipóxia reprime a transcrição de HIF1A que é revertida pelo tratamento com agente demetilante em linhagem celular de câncer de mama MDA-MB-231

Abstract

The tumor microenvironment of hypoxia is a common characteristic of solid tumors. The transcription factor HIF-1α, activated in hypoxia, coordinates the expression of genes, that make the most aggressive tumor cells. Although there is an accumulation of HIF-1α protein, in hypoxia, the HIF-1α mRNA levels are decreased. So far, little is known about the mechanisms involved in the regulation of HIF1A transcripts. Among known mechanisms that normally are associated with inhibition of transcription, the DNA methylation could elucidate a new way of control, at transcriptional level, of HIF-1α. Thereby, this study aimed to investigate the role of DNA methylation in transcriptional control HIF1A, in hypoxia. To verify the possibility of HIF1A, and also VEGFA, target gene HIF-1α, are regulated by DNA methylation, one in silico prediction of methylation sites, identified by CpG islands, was performed, in the promoter and the first exon of HIF1A and VEGFA genes, with the aid of Methprimer software. The demethylating action of 5-aza-dC was confirmed by amplifying, by PCR, of the PGR, inhibited by methylation, in breast cancer cell line, MDA-MB-231, used as an experimental model. The WST-1 assay was used to evaluate the toxicity of 5-aza-dC. The effect of 5-aza-dC on the mRNA levels of VEGFA and HIF1A were evaluated by RT-qPCR. Furthermore, alteration in the protein level of HIF-1α, after 5-aza-dC, was assessed by flow cytometry. The dose of 100 μM 5-aza-dC was used in all tests, in normoxia (48h and 72h) and hypoxia. In hypoxia, cells were treated with 5-aza-dC for 72h, with the last 24h of incubation in the hypoxia chamber. The in silico prediction identified 6 CpG islands in the assessed region of HIF1A and 3 CpG islands in VEGFA. The 5-aza-dC induced PGR expression, confirming its demethylating action and not significantly reduced cell viability. In normoxia, 5-aza-dC increased mRNA levels of HIF1A 6,5 times (± 3,8) (48h) and 2,0 times (± 0,5) (72h) and increased levels of VEGFA 2,6 (± 0,6) (48h) times and 2,4 times (± 0,5) (72h). In hypoxia, mRNA levels of HIF1A decreased by 10 (± 6,9) times while the VEGFA levels increased 1,7 (± 0,4) times. The 5-aza-dC reversed the results obtained with hypoxia, increasing mRNA levels of HIF1A 7.5 (± 2.7) times, however decreased by 1.6 (± 0.09) times the levels of VEGFA. Furthermore, the protein level of HIF-1α also increased, after 5-aza-dC, in normoxia and hypoxia. The data suggest that VEGFA and HIF1A promoters may be methylated in normoxia in a percentage still unknown. Furthermore, it is suggested that there is an increase of the degree of methylation in hypoxia, capable of inhibiting the transcription of HIF1A. However, in hypoxia, it is suggested that DNA methylation does not contribute directly to the regulation of VEGFA. Therefore, this study indicates that DNA methylation may be a new form of regulation of HIF-1α, at transcriptional level, especially in hypoxia.