RESUMO
OBJECTIVE: Functional epigenetic studies aimed to re-express transcriptionally silenced genes in renal cell carcinoma (RCC) may facilitate the ongoing search for appropriate markers supporting clinical decision-making. METHODS: The RCC cell line A-498 was treated with the DNA methyltransferase inhibitor zebularine under low-cytotoxicity conditions. RNA chip analyses revealed several upregulated transcripts that were further validated by qPCR on 49 matched pairs of human kidney tissues to identify suitable marker candidates. RESULTS: Members of the metallothionein (MT) group were remarkably downregulated in tumor tissues. MT1G and MT1H expression was decreased in 98% of cases, whereas MT2A expression was downregulated in 73% of all cases. Comparison of 308 reactivated transcripts upregulated more than 1.5-fold to published data revealed a high number of shared candidates, which supports the consistency of this experimental approach. CONCLUSION: MTs were found to be transcriptionally inactivated in human RCC. Our observations support the hypothesis of a possible involvement of these metalloproteins in renal cell carcinogenesis. Additional functional studies of these genes may provide clues for understanding renal cancers as essentially metabolic diseases.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Transformação Celular Neoplásica/genética , Epigênese Genética , Perfilação da Expressão Gênica , Neoplasias Renais/genética , Metalotioneína/genética , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Citidina/análogos & derivados , Citidina/farmacologia , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Metalotioneína/metabolismo , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição GênicaRESUMO
Transcriptional silencing, as a result of aberrant promoter hypermethylation, is a common mechanism through which genes in cancer cells become inactive. Functional epigenetic screens using demethylating agents to reexpress transcriptional silenced genes may identify such inactivated genes for needing further evaluation. We aimed to identify genes so far not known to be inactivated by promoter hypermethylation in prostate cancer. DU-145 and LNCaP cells were treated with the DNMT inhibitor zebularine. Expression changes of total RNA from treated and untreated cells were compared using an RNA expression microarray. Genes upregulated more than 2-fold were evaluated by RT-qPCR in 50 cases of paired normal and tumor tissues of prostate cancer patients. SARS was found to be downregulated in prostate cancer in 42/50 cases (84%). In addition, GADD45A and SPRY4 showed a remarkable diminished expression (88% and 74%, resp.). The gold standard for promoter hypermethylation-inactivated genes in prostate cancer (GSTP1) was repressed in 90% of our patient samples. ROC analyses reported statistically significant AUC curves in SARS, GADD45A, and GSTP1 and positive Spearman correlations were found between these genes. SARS was discovered to be a novel gene that is repressed in prostate cancer and could therefore be recommended for its involvement in prostate carcinogenesis.