Background Androgen receptor (AR) plays a crucial role as a transcription factor in promoting the development of hepatocellular carcinoma (HCC) which is prone to aberrant chromatin modifications. However, the regulatory effects of AR on epigenetic mediators in HCC remain ill-defined. Enhancer of zeste homolog 2 ( EZH2 ), an oncogene responsible for the tri-methylation of histone H3 at lysine 27 (H3K27me3), was identified to be overexpressed in approximate 70–90% of HCC cases, which prompted us to investigate whether or how AR regulates EZH2 expression. Methods Colony formation, soft agar assay, xenograft and orthotopic mouse models were used to determine cell proliferation and tumorigenicity of gene-manipulated HCC cells. Gene regulation was assessed by chromatin immunoprecipitation, luciferase reporter assay, quantitative RT-PCR and immunoblotting. Clinical relevance of candidate proteins in patient specimens was examined in terms of pathological parameters and postsurgical survival rates. Findings In this study, we found that AR upregulated EZH2 expression by binding to EZH2 promoter and stimulating its transcriptional activity. EZH2 overexpression increased H3K27me3 levels and thereby silenced the expression of Wnt signal inhibitors, resulting in activation of Wnt/β-catenin signaling and subsequently induction of cell proliferation and tumorigenesis. In a cohort of human HCC patients, concordant overexpression of AR, EZH2, H3K27me3 and active β-catenin was observed in tumor tissues compared with paired non-tumor tissues, which correlated with tumor progression and poor prognosis. These findings demonstrate a novel working model in which EZH2 mediates AR-induced Wnt/β-catenin signaling activation through epigenetic modification, and support the application of EZH2-targeted reagents for treating HCC patients.
Background Liver fibrosis is a chronic liver disease with excessive production of extracellular matrix proteins, leading to cirrhosis, hepatocellular carcinoma, and death. Purpose This study aimed at the development of a novel derivative of polyethyleneimine (PEI) that can effectively deliver transforming growth factor β (TGFβ) siRNA and inhibit chemokine receptor 4 (CXCR4) for TGFβ silencing and CXCR4 Inhibition, respectively, to treat CCl 4 -induced liver fibrosis in a mouse model. Methods Cyclam-modified PEI (PEI-Cyclam) was synthesized by incorporating cyclam moiety into PEI by nucleophilic substitution reaction. Gel electrophoresis confirmed the PEI-Cyclam polyplex formation and stability against RNAase and serum degradation. Transmission electron microscopy and zeta sizer were employed for the morphology, particle size, and zeta potential, respectively. The gene silencing and CXCR4 targeting abilities of PEI-Cyclam polyplex were evaluated by luciferase and CXCR4 redistribution assays, respectively. The histological and immunohistochemical staining determined the anti-fibrotic activity of PEI-Cyclam polyplex. The TGFβ silencing of PEI-Cyclam polyplex was authenticated by Western blotting. Results The 1 H NMR of PEI-Cyclam exhibited successful incorporation of cyclam content onto PEI. The PEI-Cyclam polyplex displayed spherical morphology, positive surface charge, and stability against RNAse and serum degradation. Cyclam modification decreased the cytotoxicity and demonstrated CXCR4 antagonistic and luciferase gene silencing efficiency. PEI-Cyclam/siTGFβ polyplexes decreased inflammation, collagen deposition, apoptosis, and cell proliferation, thus ameliorating liver fibrosis. Also, PEI-Cyclam/siTGFβ polyplex significantly downregulated α-smooth muscle actin, TGFβ, and collagen type III. Conclusion Our findings validate the feasibility of using PEI-Cyclam as a siRNA delivery vector for simultaneous TGFβ siRNA delivery and CXCR4 inhibition for the combined anti-fibrotic effects in a setting of CCl 4 -induced liver fibrosis.
Anion exchanger 1 (AE1, band 3) is a major membrane protein of red blood cells and plays a key role in acid-base homeostasis, urine acidification, red blood cell shape regulation, and removal of carbon dioxide during respiration. Though structures of the transmembrane domain (TMD) of three SLC4 transporters, including AE1, have been resolved previously in their outward-facing (OF) state, no mammalian SLC4 structure has been reported in the inward-facing (IF) conformation. Here we present the cryoEM structures of full-length bovine AE1 with its TMD captured in both IF and OF conformations. Remarkably, both IF-IF homodimers and IF-OF heterodimers were detected. The IF structures feature downward movement in the core domain with significant unexpected elongation of TM11. Molecular modeling and structure guided mutagenesis confirmed the functional significance of residues involved in TM11 elongation. Our data provide direct evidence for an elevator-like mechanism of ion transport by an SLC4 family member.
Lipid-protein interactions play an important direct role in the function of many membrane proteins. We argue they are key players in membrane structure, modulate membrane proteins in more subtle ways than direct binding, and are important for understanding the mechanism of classes of hydrophobic drugs. By directly comparing membrane proteins from different families in the same, complex lipid mixture, we found a unique lipid environment for every protein. Extending this work, we identified both differences and similarities in the lipid environment of GPCRs, dependent on which family they belong to and in some cases their conformational state, with particular emphasis on the distribution of cholesterol. More recently, we have been studying modes of coupling between protein conformation and local membrane properties using model proteins. In more applied approaches, we have used similar methods to investigate specific hypotheses on interactions of lipid and lipid-like molecules with ion channels. We conclude this perspective with some considerations for future work, including a new more sophisticated coarse-grained force field (Martini 3), an interactive visual exploration framework, and opportunities to improve sampling.
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