Induction of microRNA-127 by DNA demethylation and histone deacetylase inhbition in human cells

Abstract

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DNA methylation and histone modification play critical roles in chromatin remodeling and regulation of gene expression during mammalian development and human disease such as cancer. To analyze the distribution of DNA methylation and histone modification, we performed a chromatin scanning technique called ChAP, which couples the chromatin immunoprecipitation assay and arbitrarily primed PCR, in the human embryonic carcinoma cell line NCCIT. The ChAP assay revealed that DNMT3b and histone H3 lysine 9 methylation were mainly distributed in repetitive DNA sequences such as Alu and LINE sequences. Intriguingly, we also found that the microRNA-127 (miR-127) gene is one of the targets of both DNMT3b and histone H3 lysine 9 methylation. MicroRNAs (miRNAs) are 21 to 23 nucleotides long non-coding RNAs that regulate gene expression by repressing translation or by directing mRNA degradation. These miRNAs are considered to be involved in development of animals. Although expression of mouse miR-127 has been verified in mouse brain and spleen, expression of miR-127 has never been verified in humans. We found that the miR-127 gene was unmethylated in human sperm, methylated about 50% in human testis and completely methylated in all the other human tissues studied. After treatment with 5-aza-2’-deoxycytidine (5-aza-CdR), the average methylation level of the miR-127 gene was significantly decreased in T24 cells. Moreover, Northern blot analysis of miR-127 in T24 cells treated with 5-aza-CdR and/or histone deacetylase (HDAC) inhibitor, phenylbutyric acid (PBA) demonstrated that neither 5-aza-CdR or PBA alone induced miR-127; only the combination treatment of 5-aza-CdR and PBA could significantly induce expression of miR-127. This is the first report that reveals that a miRNA is inducible by DNA demethylation and histone deacetylase inhibition, and also miR-127 was detected in human cells for the first time. These results strongly suggest a novel effect of epigenetic therapy that couples DNA methylation and HDAC inhibitors to induce expression of miRNAs. These miRNAs can potentially regulate gene expression of target genes that are important in human tumorigenesis.