New advances of DNA methylation in liver fibrosis, with special emphasis on the crosstalk between microRNAs and DNA methylation machinery

AbstractEpigenetics refers to the study of heritable changes in the pattern of gene expression that is controlled by a mechanism specifically not due to changes the primary DNA sequence. Well-known epigenetic mechanisms include DNA methylation, post-translational histone modifications and RNA-based mechanisms including those controlled by small non-coding RNAs (miRNAs). Recent studies have shown that epigenetic modifications orchestrate the hepatic stellate cell (HSC) activation and liver fibrosis. In this review we focus on the aberrant methylation of CpG island promoters of select genes is the prominent epigenetic mechanism to effectively silence gene transcription facilitating HSC activation and liver fibrosis. Furthermore, we also discuss epigenetic dysregulation of tumor-suppressor miRNA genes by promoter DNA methylation and the interaction of DNA methylation with miRNAs involved in the regulation of HSC activation and liver fibrosis. Recent advances in epigenetics alterations in the pathogenesis of liver fibrosis and their possible use as new therapeutic targets and biomarkers

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Melittin restores PTEN expression by down-regulating HDAC2 in human hepatocelluar carcinoma HepG2 cells.

Melittin is a water-soluble toxic peptide derived from the venom of the bee. Although many studies show the anti-tumor activity of melittin in human cancer including glioma cells, the underlying mechanisms remain elusive. Here the effect of melittin on human hepatocelluar carcinoma HepG2 cell proliferation in vitro and further mechanisms was investigated. We found melittin could inhibit cell proliferation in vitro using Flow cytometry and MTT method. Besides, we discovered that melittin significantly downregulated the expressions of CyclinD1 and CDK4. Results of western Blot and Real-time PCR analysis indicated that melittin was capable to upregulate the expression of PTEN and attenuate histone deacetylase 2 (HDAC2) expression. Further studies demonstrated that knockdown of HDAC2 completely mimicked the effects of melittin on PTEN gene expression. Conversely, it was that the potential utility of melittin on PTEN expression was reversed in cells treated with a recombinant pEGFP-C2-HDAC2 plasmid. In addition, treatment with melittin caused a downregulation of Akt phosphorylation, while overexpression of HDAC2 promoted Akt phosphorylation. These findings suggested that the inhibitory of cell growth by melittin might be led by HDAC2-mediated PTEN upregulation, Akt inactivation, and inhibition of the PI3K/Akt signaling pathways

Effects of PTEN on Akt activation in HepG2 cells.

<p>HepG2 cells were treated with 1, 4, 8 µg/ml of melittin and 0.5 µmol/ml TSA for 24 h. The p-Akt and Akt proteins expression were analyzed by Western blot (a). *P<0.05, **P<0.01 compared to cells without melittin treatment. Silencing of HDAC2 genes in HepG2 cells was performed and phosphorylation of Akt was checked by Western blot (b) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095520#s2" target="_blank">Materials and methods</a> section. Representative images of at least three independent experiments are shown and results of densitometric analyses (mean±SE) are shown. **P<0.01 vs controls and scrambled siRNA. HepG2 cells were treated with pEGFP-C2, pEGFP-C2-HDAC2 plasmid, pEGFP-C2-HDAC2 plasmid and melittin, pEGFP-C2-HDAC2 plasmid and TSA for 24 h. Phosphorylation of Akt was checked by Western blot (c). **P<0.01 vs control and vector group. ^#P<0.05 vs pEGFP-C2-HDAC-2 plasmid group.</p

Effect of melittin on the expression of CyclinD1 and CDK4 in HepG2 cells.

<p>(a and b)Total cellular proteins and RNA were prepared and the expressions of CyclinD1 and CDK4 proteins and mRNA were analyzed using Western blot and RT-PCR. β-actin was used as an internal control. Representative blots and images of three independent experiments are shown. *P<0.05, **P<0.01 vs Control. Statistical analysis was performed by ANOVA.</p

The sequences of siRNA Oligo.

<p>The sequences of siRNA Oligo.</p

HDAC2 knockdown mimicked the effect of melittin in HepG2 cells.

<p>HepG2 cells were transfected with HDAC2 siRNA for 24 h. The HDAC2 and PTEN mRNA expressions were analyzed by RT-PCR (a). The HDAC2, PTEN and ac-H3 proteins expression was analyzed by Western blot (b) and (c). Representative images from 3 to 4 independent experiments are shown. **P<0.01 vs control and scrambled siRNA.</p

Effect of pEGFP-C2-HDAC2 plasmid on PTEN in HepG2 cells.

<p>HepG2 cells were treated with pEGFP-C2, pEGFP-C2-HDAC2 plasmid, pEGFP-C2-HDAC2 plasmid and melittin, pEGFP-C2-HDAC2 plasmid and TSA for 24 h. The PTEN mRNA expression was analyzed by real-time PCR (a). The PTEN protein expression was analyzed by Western blot (b). Relative PTEN mRNA and protein levels are presented as mean ± standard of the mean (S.E.) of optical densities from three separated experiments. **P<0.01 vs control and vector. ^#P<0.05 vs pEGFP-C2-HDAC2 plasmid group.</p

The mechanism of melittin inhibits liver cancer cell proliferation.

<p>Melittin downregulates the expression of HDAC2. Then, inhibition of HDAC2 leads to an increased levels of PTEN and promotes Akt inactivation, inhibition of the PI3K/Akt signaling pathways, thereby inhibits cell proliferation.</p