RESUMO
This study aimed to clarify epigenetic and genetic alterations that occur during renal carcinogenesis. The original method includes chromosome 3 specific NotI-microarrays containing 180 NotI-clones associated with 188 genes for hybridization with 23 paired normal/tumor DNA samples of primary clear cell renal cell carcinomas (ccRCC). Twenty-two genes showed methylation and/or deletion in 17-57% of tumors. These genes include tumor suppressors or candidates (VHL, CTDSPL, LRRC3B, ALDH1L1, and EPHB1) and genes that were not previously considered as cancer-associated (e.g., LRRN1, GORASP1, FGD5, and PLCL2). Bisulfite sequencing analysis confirmed methylation as a frequent event in ccRCC. A set of six markers (NKIRAS1/RPL15, LRRN1, LRRC3B, CTDSPL, GORASP1/TTC21A, and VHL) was suggested for ccRCC detection in renal biopsies. The mRNA level decrease was shown for 6 NotI-associated genes in ccRCC using quantitative PCR: LRRN1, GORASP1, FOXP1, FGD5, PLCL2, and ALDH1L1. The majority of examined genes showed distinct expression profiles in ccRCC and papillary RCC. The strongest extent and frequency of downregulation were shown for ALDH1L1 gene both in ccRCC and papillary RCC. Moreover, the extent of ALDH1L1 mRNA level decrease was more pronounced in both histological types of RCC stage III compared with stages I and II (P = 0.03). The same was observed for FGD5 gene in ccRCC (P < 0.06). Dedicated to thememory of Eugene R. Zabarovsky.
Assuntos
Carcinoma de Células Renais/genética , Cromossomos Humanos Par 3/genética , Epigênese Genética/genética , Neoplasias Renais/genética , Proteínas de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Deleção Cromossômica , Marcadores Genéticos/genética , Variação Genética/genética , HumanosRESUMO
BACKGROUND: Large-scale screening methods are widely used to reveal cancer-specific DNA methylation markers. We previously identified non-satellite 3.3-kb repeats associated with facioscapulohumeral muscular dystrophy (FSHD) as hypermethylated in cervical cancer in genome-wide screening. To determine whether hypermethylation of 3.3-kb repeats is a tumor-specific event and to evaluate frequency of this event in tumors, we investigated the 3.3-kb repeat methylation status in human papilloma virus (HPV)-positive cervical tumors, cancer cell lines, and normal cervical tissues. Open reading frames encoding DUX family proteins are contained within some 3.3-kb repeat units. The DUX mRNA expression profile was also studied in these tissues. METHODS: The methylation status of 3.3-kb repeats was evaluated by Southern blot hybridization and bisulfite genomic sequencing. The expression of DUX mRNA was analyzed by RT-PCR and specificity of PCR products was confirmed by sequencing analysis. RESULTS: Hypermethylation of 3.3-kb repeats relative to normal tissues was revealed for the first time in more than 50% (18/34) of cervical tumors and in 4 HPV-positive cervical cancer cell lines. Hypermethylation of 3.3-kb repeats was observed in tumors concurrently with or independently of hypomethylation of classical satellite 2 sequences (Sat2) that were hypomethylated in 75% (15/20) of cervical tumors. We have revealed the presence of transcripts highly homologous to DUX4 and DUX10 genes in normal tissues and down-regulation of transcripts in 68% of tumors with and without 3.3-kb repeats hypermethylation. CONCLUSION: Our results demonstrate that hypermethylation rather than hypomethylation of 3.3-kb repeats is the predominant event in HPV-associated cervical cancer and provide new insight into the epigenetic changes of repetitive DNA elements in carcinogenesis.