RESUMEN
Mitochondrial DNA mutations have been shown to play important roles in the pathogenesis of hepatocellular carcinoma (HCC). In particular, genes encoding mitochondrial tRNA (mt-tRNA) are hotspots for pathogenic mutations associated with HCC. Recently, an increasing number of studies have reported the involvement of such mutations in this disease. As a result, several mt-tRNA mutations associated with HCC have been described. Some of these are neutral polymorphisms and may not cause mitochondrial dysfunction. Moreover, the molecular mechanisms by which these pathogenic mutations result in HCC remain unclear. To address this problem, we evaluated five mt-tRNA variants (tRNA(Val) T1659C, tRNA(Ala) G5650A, tRNA(Arg) T10463C, tRNA(Glu) A14679G, and tRNA(Pro) C15975T) implicated in the clinical manifestation of HCC in humans. We performed evolutionary conservation analysis and used a bioinformatic tool to predict the secondary structure of the mt-tRNAs carrying these mutations. Using an established pathogenicity scoring system, we classified T10463C and A14679G as neutral polymorphisms, and determined that the T1659C, G5650A, and C15975T variants should be regarded as pathogenic mutations. To the best of our knowledge, this is the first report to establish the pathogenicity of HCC-associated mt-tRNA mutations.
Asunto(s)
Carcinoma Hepatocelular/genética , ADN Mitocondrial/genética , Neoplasias Hepáticas/genética , Tasa de Mutación , Polimorfismo de Nucleótido Simple , Humanos , ARN de Transferencia/genéticaRESUMEN
DNA hypomethylation may activate oncogene transcription, thus promoting carcinogenesis and tumor development. S-adenosylmethionine (SAM) is a methyl donor in numerous methylation reactions and acts as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA. The main objectives of this study were to determine whether DNA hypomethylation correlated with vascular endothelial growth factor-C (VEGF-C) expression, and the effect of SAM on VEGF-C methylation and gastric cancer growth inhibition. VEGF-C expression was assayed by Western blotting and RT-qPCR in gastric cancer cells, and by immunohistochemistry in tumor xenografts. VEGF-C methylation was assayed by bisulfite DNA sequencing. The effect of SAM on cell apoptosis was assayed by flow cytometry analyses and its effect on cancer growth was assessed in nude mice. The VEGF-C promoters of MGC-803, BGC-823, and SGC-7901 gastric cancer cells, which normally express VEGF-C, were nearly unmethylated. After SAM treatment, the VEGF-C promoters in these cells were highly methylated and VEGF-C expression was downregulated. SAM also significantly inhibited tumor growth in vitro and in vivo. DNA methylation regulates expression of VEGF-C. SAM can effectively induce VEGF-C methylation, reduce the expression of VEGF-C, and inhibit tumor growth. SAM has potential as a drug therapy to silence oncogenes and block the progression of gastric cancer.
Asunto(s)
Animales , Humanos , Masculino , Antineoplásicos/farmacología , Metilación de ADN/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , S-Adenosilmetionina/farmacología , Neoplasias Gástricas/tratamiento farmacológico , Factor C de Crecimiento Endotelial Vascular/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Carcinogénesis/efectos de los fármacos , Metilación de ADN/genética , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/fisiología , Xenoinjertos/efectos de los fármacos , Inmunohistoquímica , Ratones Desnudos , Oncogenes/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , ARN Mensajero/análisis , Neoplasias Gástricas/metabolismo , Factor C de Crecimiento Endotelial Vascular/efectos de los fármacos , Factor C de Crecimiento Endotelial Vascular/genéticaRESUMEN
DNA hypomethylation may activate oncogene transcription, thus promoting carcinogenesis and tumor development. S-adenosylmethionine (SAM) is a methyl donor in numerous methylation reactions and acts as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA. The main objectives of this study were to determine whether DNA hypomethylation correlated with vascular endothelial growth factor-C (VEGF-C) expression, and the effect of SAM on VEGF-C methylation and gastric cancer growth inhibition. VEGF-C expression was assayed by Western blotting and RT-qPCR in gastric cancer cells, and by immunohistochemistry in tumor xenografts. VEGF-C methylation was assayed by bisulfite DNA sequencing. The effect of SAM on cell apoptosis was assayed by flow cytometry analyses and its effect on cancer growth was assessed in nude mice. The VEGF-C promoters of MGC-803, BGC-823, and SGC-7901 gastric cancer cells, which normally express VEGF-C, were nearly unmethylated. After SAM treatment, the VEGF-C promoters in these cells were highly methylated and VEGF-C expression was downregulated. SAM also significantly inhibited tumor growth in vitro and in vivo. DNA methylation regulates expression of VEGF-C. SAM can effectively induce VEGF-C methylation, reduce the expression of VEGF-C, and inhibit tumor growth. SAM has potential as a drug therapy to silence oncogenes and block the progression of gastric cancer.