Epigenetic Effects of Resveratrol on Oncogenic Signaling in Breast Cancer.

The crosstalk between oncogenic signaling pathways plays a crucial role in driving cancer development. We previously demonstrated that dietary polyphenols, specifically resveratrol (RSV) and other stilbenoids, epigenetically target oncogenes for silencing via DNA hypermethylation in breast cancer. In the present study, we identify signal transduction regulators among RSV-hypermethylated targets and investigate the functional role of RSV-mediated DNA hypermethylation in the regulation of Hedgehog and Wnt signaling. Non-invasive ER-positive MCF-7 and highly invasive triple-negative MCF10CA1a human breast cancer cell lines were used as experimental models. Upon 9-day exposure to 15 µM RSV, pyrosequencing and qRT-PCR were performed to assess DNA methylation and expression of GLI2 and WNT4, which are upstream regulators of the Hedgehog and Wnt pathways, respectively. Our results showed that RSV led to a DNA methylation increase within GLI2 and WNT4 enhancers, which was accompanied by decreases in gene expression. Consistently, we observed the downregulation of genes downstream of the Hedgehog and Wnt signaling, including common targets shared by both pathways, CCND1 and CYR61. Further analysis using chromatin immunoprecipitation identified increased H3K27 trimethylation and decreased H3K9 and H3K27 acetylation, along with abolishing OCT1 transcription factor binding. Those changes indicate a transcriptionally silent chromatin state at GLI2 and WNT4 enhancers. The inhibition of the Wnt signal transduction was confirmed using a phospho-antibody array that demonstrated suppression of positive and stimulation of negative Wnt regulators. In conclusion, our results provide scientific evidence for dietary polyphenols as epigenetics-modulating agents that act to re-methylate and silence oncogenes, reducing the oncogenic signal transduction. Targeting such an action could be an effective strategy in breast cancer prevention and/or adjuvant therapy.

Novel database for accA gene revealed a vertical variability pattern of autotrophic carbon fixation potential of ammonia oxidizing archaea in a permeable subterranean estuary.

The autotrophic carbon fixation pathway of ammonia-oxidizing archaea (AOA) was the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle, of which the acetyl-CoA carboxylase α-submit (accA) gene is widely recognized as the indicator. To date, there is no reference database or suitable cut-off value for operational taxonomic unit (OTU) clustering to analyze the diversity of AOA based on the accA gene. In this study, a reference database with 489 sequences was constructed, all the accA gene sequences was obtained from the AOA enrichment culture, pure culture and environmental samples. Additionally, the 79% was determined as the cut-off value for OTU clustering by comparing the similarity between the accA gene and the 16S rRNA gene. The developed method was verified by analyzing samples from the subterranean estuary and a vertical variation pattern of autotrophic carbon fixation potential of AOA was revealed. This study provided an effective method to analyze the diversity and autotrophic carbon fixation potential of AOA based on accA gene.

DNA hypermethylation of Kiss1r promoter and reduction of hepatic Kiss1r in female rats with type 2 diabetes.

Kisspeptin, produced from the brain and peripheral tissues, may constitute an important link in metabolic regulation in response to external cues, such as diet. The kisspeptin system is well described in the brain. However, its function and regulation in the peripheral tissues, especially in relation to metabolic disease and sex differences, remain to be elucidated. As Kiss1 and Kiss1r, encoding for kisspeptin and kisspeptin receptors, respectively, are altered by overnutrition/fasting and regulated by DNA methylation during puberty and cancer, epigenetic mechanisms in metabolic disorders are highly probable. In the present study, we experimentally induced type 2 diabetes mellitus (DM2) in female Wistar rats using high-fat diet/streptozocin. We analysed expression and DNA methylation of Kiss1 and Kiss1r in the peripheral tissues, using quantitative-reverse-transcription PCR (qRT-PCR) and pyrosequencing. We discovered differential expression of Kiss1 and Kiss1r in peripheral organs in DM2 females, as compared with healthy controls, and the profile differed from patterns reported earlier in males. DM2 in females was linked to the increased Kiss1 mRNA in the liver and increased Kiss1r mRNA in the liver and adipose tissue. However, Kiss1r promoter was hypermethylated in the liver, suggesting gene silencing. Indeed, the increase in DNA methylation of Kiss1r promoter was accompanied by a reduction in Kiss1r protein, implying epigenetic or translational gene repression. Our results deliver novel evidence for tissue-specific differences in Kiss1 and Kiss1r expression in peripheral organs in DM2 females and suggest DNA methylation as a player in regulation of the hepatic kisspeptin system in DM2.

Epigenetic aberrations of gene expression in a rat model of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is mostly triggered by environmental and life-style factors and may involve epigenetic aberrations. However, a comprehensive documentation of the link between the dysregulated epigenome, transcriptome, and liver carcinogenesis is lacking. In the present study, Fischer-344 rats were fed a choline-deficient (CDAA, cancer group) or choline-sufficient (CSAA, healthy group) L-amino acid-defined diet. At the end of 52 weeks, transcriptomic alterations in livers of rats with HCC tumours and healthy livers were investigated by RNA sequencing. DNA methylation and gene expression were assessed by pyrosequencing and quantitative reverse-transcription PCR (qRT-PCR), respectively. We discovered 1,848 genes that were significantly differentially expressed in livers of rats with HCC tumours (CDAA) as compared with healthy livers (CSAA). Upregulated genes in the CDAA group were associated with cancer-related functions, whereas macronutrient metabolic processes were enriched by downregulated genes. Changes of highest magnitude were detected in numerous upregulated genes that govern key oncogenic signalling pathways, including Notch, Wnt, Hedgehog, and extracellular matrix degradation. We further detected perturbations in DNA methylating and demethylating enzymes, which was reflected in decreased global DNA methylation and increased global DNA hydroxymethylation. Four selected upregulated candidates, Mmp12, Jag1, Wnt4, and Smo, demonstrated promoter hypomethylation with the most profound decrease in Mmp12. MMP12 was also strongly overexpressed and hypomethylated in human HCC HepG2 cells as compared with primary hepatocytes, which coincided with binding of Ten-eleven translocation 1 (TET1). Our findings provide comprehensive evidence for gene expression changes and dysregulated epigenome in HCC pathogenesis, potentially revealing novel targets for HCC prevention/treatment.