Eventos epigenéticos no ameloblastoma: um enfoque na metilação e transcrição de metaloproteinases da matriz e perda de heterozigosidade do gene PTCH

Abstract

Ameloblastoma is a benign odontogenic neoplasm with an aggressive behavior and high recurrence rates. Some studies have been performed to elucidated molecular mechanisms and genetic alterations evolving tumor pathogenesis. Increased expression of matrix metalloproteinases (MMPs) is reported in ameloblastomas. Alterations in the Hedgehog signalling pathway, including PTCH gene mutations, also have been associated with the pathogenesis of some odontogenic tumours. In the present study, we focalizes two purposes of search related to methylation of matrix metalloproteinasis and heterozygosity loss (LOH) in the chromosome locus of PTCH gene. In the first, we hypothesized that epigenetic alterations may regulate MMP expression in ameloblastomas. Therefore, we investigated the MMP-2 and MMP-9 genes methylation status together with mRNA transcription and protein expression in ameloblastoma. Methylation-specific polymerase chain reaction (MSP-PCR) and methylation analysis by restriction enzyme was performed to evaluate methylation profile of MMP-2 and MMP-9 genes in 12 ameloblastoma samples, 12 dental follicles and 12 healthy gingiva fragments, included as controls. Furthermore, we investigated the transcription levels of the genes by quantitative reverse-transcription PCR (qRT-PCR). Zymography was performed to verify protein expression. Ameloblastoma and dental follicle showed a high frequency of unmethylated MMP-2 and MMP-9, whereas healthy gingival samples presented a sharp prevalence of methylated MMPs. Dental follicles showed a significantly higher MMP-2 mRNA expression than ameloblastomas and healthy gingiva. However, higher expression levels of MMP-9 were found in ameloblastomas than in dental follicles and healthy gingiva. All ameloblastomas showed positive expression of MMP-2 and MMP-9 proteins. To other purpose of study, we assess LOH at the PTCH locus in ameloblastoma. Twelve ameloblastomas were included and LOH was assessed by using 3 microsatellite markers D9S252, D9S127, and D9S287, and 3 single-nucleotide polymorphisms (SNPs) rs112794371, rs111446700, rs357564 all located at the PTCH gene locus. Furthermore, we investigated GLI1 and GLI2 transcription levels by quantitative reverse-transcription PCR (qRT-PCR) in 8 ameloblastomas and concomitantly, PTCH protein levels by immunohistochemical analysis. LOH at 9q21.33-9q.31 was detected in 4/10 (40.0%) informative cases of ameloblastoma. All 8 analyzed samples expressed GLI1 mRNA and seven cases GLI2 mRNA. Interestingly, LOH at the PTCH locus was not correlated with GLI1 or GLI2 transcription levels, nor was there any correlation with PTCH protein expression. Considering all results, our findings point to an increased expression of MMP-9 in ameloblastoma, possibly modulated by methylation of the gene. Furthermore, we suggested that LOH in the PTCH region may be relevant to the pathogenesis of ameloblastoma, but may target a different gene than PTCH. All together, these can be new insights that need to be more explored to understand pathogenesis of ameloblastoma.