Análise de metilação em diferentes regiões de genes supressores de tumor no carcinoma de células escamosas bucal
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Data
2020-05-04
Autores
Miranda, Keila Cristina
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Universidade Estadual Paulista (Unesp)
Resumo
O carcinoma de células escamosas bucal (CCEB) apresenta alterações epigenéticas, como a metilação de DNA, na região promotora de genes supressores de tumor (GST), mas pouco se sabe em relação a outras regiões gênicas. Este estudo teve como objetivo investigar o perfil de metilação de diferentes regiões de um grupo de GSTs, validar dados significativos por meio de análises in silico e experimental, e correlacionar estes achados aos dados clínico-patológicos dos pacientes. Na plataforma HumanMethylation450 Beadchip, foram utilizadas 53 amostras de CCEB e 53 de tecidos não-neoplásicos adjacentes ao tumor (TA). O nível de metilação destes tecidos foram comparados considerando Δβ ≤ -0,10 ou ≥ 0,10, p < 0,01 e FDR < 0,01. Os resultados obtidos foram validados no TCGA e por pirosequenciamento utilizando 15 amostras independentes de CCEB e 15 TA. Os genes BRCA1, FHIT, MGMT, RASSF1 e RUNX3 apresentaram sítios hipermetilados na região promotora, 5’UTR e/ou 1º éxon, enquanto CDH13, DAPK1, HOXA1, PDLIM4, RARB, TIMP3, TP73 e VHL mostraram hipermetilação no corpo gênico e/ou 3’UTR. Foram identificados sítios diferencialmente metilados entre o CCEB e TA para os genes BRCA1, FHIT e HOXA1. Na validação experimental, apenas o sítio CpG na região 3’UTR do gene HOXA1 mostrou diferença significativa (p < 0,01) entre o CCEB e TA, 55,5% e 36,9% respectivamente, estando sua metilação e maior expressão (FC = 10,58) positivamente correlacionada à carga tabágica (p = 0,04) e recidiva (p = 0,03). A análise in silico utilizada mostrou-se efetiva na identificação dos alvos e comparação entre os grupos. A metilação de diferentes regiões de genes supressores de tumor é um evento frequente no CCEB, entretanto pouca diferença é encontrada entre o perfil de metilação tumoral e as margens da lesão. A metilação do sítio CpG na região 3’UTR do HOXA1 em CCEB está correlacionada a expressão aberrante deste gene e maiores episódios de recorrência.
Oral squamous cell carcinoma (OSCC) presents epigenetic changes, such as DNA methylation, in tumor suppressor genes (GST) promoter regions, but little is known about other gene regions. This study aimed to investigate the methylation profile of different regions of a group of GSTs, to validate significant data through in silico and experimental analyzes, and to correlate these findings with the patients' clinical-pathological data. In the HumanMethylation450 Beadchip platform, 53 samples of CCEB and 53 of non-neoplastic tissues adjacent to the tumor (AT) were used. The methylation level of these tissues was compared considering ∆β ≤ -0.10 or ≥ 0.10, p <0.01 and FDR < 0.01. The results obtained were validated at TCGA and by pyrosquencing using 15 independent samples of CCEB and 15 TA. The BRCA1, FHIT, MGMT, RASSF1 and RUNX3 genes showed hypermethylated sites in the promoter region, 5'UTR and / or 1st exon, while CDH13, DAPK1, HOXA1, PDLIM4, RARB, TIMP3, TP73 and VHL showed hypermethylation in the gene body and / or 3'UTR. Differently methylated sites were identified between the OSCC and AT for the BRCA1, FHIT and HOXA1 genes. In experimental validation, only the CpG site in the 3'UTR region of the HOXA1 gene showed a significant difference (p < 0.01) between OSCC and AT, 55.5% and 36.9% respectively, with its methylation and greater expression ( FC = 10.58) positively correlated with smoking burden (p = 0.04) and recurrence (p = 0.03). The in silico analysis used was effective in identifying the targets and comparing the groups. Methylation of different regions of GST is a frequent event in the OSCC, however little difference is found between the tumor methylation profile and the lesion margins. Methylation of the CpG site in the 3'UTR region of HOXA1 in CCEB is correlated with the aberrant expression of this gene and with greater recurrence episodes.
Oral squamous cell carcinoma (OSCC) presents epigenetic changes, such as DNA methylation, in tumor suppressor genes (GST) promoter regions, but little is known about other gene regions. This study aimed to investigate the methylation profile of different regions of a group of GSTs, to validate significant data through in silico and experimental analyzes, and to correlate these findings with the patients' clinical-pathological data. In the HumanMethylation450 Beadchip platform, 53 samples of CCEB and 53 of non-neoplastic tissues adjacent to the tumor (AT) were used. The methylation level of these tissues was compared considering ∆β ≤ -0.10 or ≥ 0.10, p <0.01 and FDR < 0.01. The results obtained were validated at TCGA and by pyrosquencing using 15 independent samples of CCEB and 15 TA. The BRCA1, FHIT, MGMT, RASSF1 and RUNX3 genes showed hypermethylated sites in the promoter region, 5'UTR and / or 1st exon, while CDH13, DAPK1, HOXA1, PDLIM4, RARB, TIMP3, TP73 and VHL showed hypermethylation in the gene body and / or 3'UTR. Differently methylated sites were identified between the OSCC and AT for the BRCA1, FHIT and HOXA1 genes. In experimental validation, only the CpG site in the 3'UTR region of the HOXA1 gene showed a significant difference (p < 0.01) between OSCC and AT, 55.5% and 36.9% respectively, with its methylation and greater expression ( FC = 10.58) positively correlated with smoking burden (p = 0.04) and recurrence (p = 0.03). The in silico analysis used was effective in identifying the targets and comparing the groups. Methylation of different regions of GST is a frequent event in the OSCC, however little difference is found between the tumor methylation profile and the lesion margins. Methylation of the CpG site in the 3'UTR region of HOXA1 in CCEB is correlated with the aberrant expression of this gene and with greater recurrence episodes.
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Palavras-chave
Metilação do DNA, DNA methylation, Epigenética, Epigenetic, Ilhas CpG, CpG island, Câncer oral, Oral cancer