Avaliação de mecanismos de regulação de SPRR3 em carcinoma epidermoide de esôfago

Abstract

Esophageal cancer (EC) is the eighth most frequent tumor and represents a sixth cancer-related death cause, with esophageal squamous cell carcinoma (ESCC) accounting for 80% of cases. SPRR3 is a protein associated with terminal epithelial differentiation and a gradual loss of both SPRR3 gene and protein expression in the malignant transformation of healthy esophagus into ESCC has been seen in some studies. However, studies on the molecular mechanisms involved in the regulation of SPRR3 are limited. Therefore, the objective of the study was to investigate possible mechanisms related to the regulation of this gene. Initially, after identifying the decrease of SPRR3 gene expression in 35 ESCC samples in comparision to their respective tumor surrounding mucosa, we evaluated DNA methylation levels in 3 gene regions (A, B and C) by pyrosquencing in these samples. The results showed a negative correlation between methylation levels of SPRR3 and its gene expression in the 3 regions analyzed. Then, we evaluated an expression of the three major DNA methyl transferases (DNMTs), enzymes involved in DNA methylation process, using the same group of samples, and we observed correlation between expression of DNMT1, DNMT3A and DNMT3B with genetic expression and methylation levels of SPRR3, with the exception of DNMT3A and site C. After this, we performed the treatment of ESCC cell lines (OE21, TE1, TE13) with DNMT inhibitor (decitabine). Treatment with decitabine induced cell demethylation and increased SPRR3 gene expression. Next, we evaluated the involvement of miRNAs in the silencing of SPRR3 and, analyzing data deposited in the TCGA database, we identified that miR-21 and miR-503 presented an inverse correlation with SPRR3 expression. MiR-503 has SPRR3 as the predicted target, while miR-21 appears to act indirectly, according to predictive analysis using MiRWalk site. Therefore, our results suggest a direct regulation by miR-503 and an indirect action of miR-21 on the silencing of SPPR3. This study also identified, through in silico analysis using the MatInspector and JASPAR programs, 24 transcription factors that could affect the regulation of SPRR3 expression. Among these, 13 genes showed a significant correlation with SPRR3 expression in ESCC samples. We identified the transcriptional factor MEIS1 as a possible target of miR-21 and that it could act in the regulation of SPRR3. Thus, our data together suggest that epigenetic mechanisms are involved in the silencing of SPRR3 in ESCC. As conclusion, we can relate the reduction of SPRR3 expression in ESCC with DNA methylation mediated by DNMTs, as well as we can suggest the involvement of other mechanisms in the silencing of this gene, such as the influence by transcription factors and the direct and indirect action of miRNAs