Background: Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been linked with proliferation, survival, invasion and angiogenesis of a variety of human cancer cells, including hepatocellular carcinoma (HCC). Thus, novel agents that can suppress STAT3 activation have potential for both prevention and treatment of HCC. Here we report, garcinol, a polyisoprenylated benzophenone, could suppress STAT3 activation in HCC cell lines and in xenografted tumor of HCC in nude mice model.Experimental design: Different HCC cell lines have been treated with garcinol and the inhibition of STAT3 activation, dimerization and acetylation have been checked by immunoblotting, immuno-fluorescence, and DNA binding assays. Xenografted tumor model has been generated in nude mice using HCC cell line and effect of garcinol in the inhibition of tumor growth has been investigated.Results: Garcinol could inhibit both constitutive and interleukin (IL-6) inducible STAT3 activation in HCC cells. Computational modeling showed that garcinol could bind to the SH2 domain of STAT3 and suppress its dimerizationin vitro. Being an acetyltransferase inhibitor, garcinol also inhibits STAT3 acetylation and thus impairs its DNA binding ability. The inhibition of STAT3 activation by garcinol led to the suppression of expression of various genes involved in proliferation, survival, and angiogenesis. It also suppressed proliferation and induced substantial apoptosis in HCC cells. Remarkably, garcinol inhibited the growth of human HCC xenograft tumors in athymic nu/nu mice, through the inhibition of STAT3 activation.Conclusion: Overall, our results suggest that garcinol exerts its anti-proliferative and pro-apoptotic effects through suppression of STAT3 signaling in HCC bothin vitroandin vivo.

Rationale:: AngII (angiotensin II)-mediated vascular smooth muscle cell (VSMC) dysfunction plays a major role in hypertension. Long noncoding RNAs have elicited much interest, but their molecular roles in AngII actions and hypertension are unclear. Objective:: To investigate the regulation and functions of a novel long noncoding RNA growth factor– and proinflammatory cytokine–induced vascular cell-expressed RNA ( Giver ), in AngII-mediated VSMC dysfunction. Methods and Results:: RNA-sequencing and real-time quantitative polymerase chain reactions revealed that treatment of rat VSMC with AngII increased the expression of Giver and Nr4a3 , an adjacent gene encoding a nuclear receptor. Similar changes were observed in rat and mouse aortas treated ex vivo with AngII. RNA-FISH (fluorescence in situ hybridization) and subcellular fractionation showed predominantly nuclear localization of Giver . AngII increased Giver expression via recruitment of Nr4a3 to Giver promoter. Microarray profiling and real-time quantitative polymerase chain reaction validation in VSMC showed that Giver knockdown attenuated the expression of genes involved in oxidative stress ( Nox1 ) and inflammation ( Il6 , Ccl2 , Tnf ) but increased Nr4a3 . Conversely, endogenous Giver overexpression showed opposite effects supporting its role in oxidative stress and inflammation. Chromatin immunoprecipitation assays showed Giver overexpression also increased Pol II (RNA polymerase II) enrichment and decreased repressive histone modification histone H3 trimethylation on lysine 27 at Nox1 and inflammatory gene promoters. Accordingly, Giver knockdown inhibited AngII-induced oxidative stress and proliferation in rat VSMC. RNA-pulldown combined with mass spectrometry showed Giver interacts with nuclear and chromatin remodeling proteins and corepressors, including NONO (non-pou domain-containing octamer-binding protein). Moreover, NONO knockdown elicited similar effects as Giver knockdown on the expression of key Giver -regulated genes. Notably, GIVER and NR4A3 were increased in AngII-treated human VSMC and in arteries from hypertensive patients but attenuated in hypertensive patients treated with ACE (angiotensin-converting enzyme) inhibitors or angiotensin receptor blockers. Furthermore, human GIVER also exhibits partial functional conservation with rat Giver . Conclusions:: Giver and its regulator Nr4a3 are important players in AngII-mediated VSMC dysfunction and could be novel targets for antihypertensive therapy.

Objective—: Macrophages play key roles in inflammation and diabetic vascular complications. Emerging evidence implicates long noncoding RNAs in inflammation, but their role in macrophage dysfunction associated with inflammatory diabetic complications is unclear and was therefore investigated in this study. Approach and Results—: RNA-sequencing and real-time quantitative PCR demonstrated that a long noncoding RNA Dnm3os (dynamin 3 opposite strand) is upregulated in bone marrow-derived macrophages from type 2 diabetic db/db mice, diet-induced insulin-resistant mice, and diabetic ApoE ^−/− mice, as well as in monocytes from type 2 diabetic patients relative to controls. Diabetic conditions (high glucose and palmitic acid) induced Dnm3os in mouse and human macrophages. Promoter reporter analysis and chromatin immunoprecipitation assays demonstrated that diabetic conditions induce Dnm3os via NF-κB activation. RNA fluorescence in situ hybridization and real-time quantitative PCRs of subcellular fractions demonstrated nuclear localization and chromatin enrichment of Dnm3os in macrophages. Stable overexpression of Dnm3os in macrophages altered global histone modifications and upregulated inflammation and immune response genes and phagocytosis. Conversely, RNAi-mediated knockdown of Dnm3os attenuated these responses. RNA pull-down assays with macrophage nuclear lysates identified nucleolin and ILF-2 (interleukin enhancer-binding factor 2) as protein binding partners of Dnm3os , which was further confirmed by RNA fluorescence in situ hybridization immunofluorescence. Furthermore, nucleolin levels were decreased in diabetic conditions, and its knockdown enhanced Dnm3os -induced inflammatory gene expression and histone H3K9-acetylation at their promoters. Conclusions—: These results demonstrate novel mechanisms involving upregulation of long noncoding RNA Dnm3os , disruption of its interaction with nucleolin, and epigenetic modifications at target genes that promote macrophage inflammatory phenotype in diabetes mellitus. The data could lead to long noncoding RNA-based therapies for inflammatory diabetes mellitus complications.

Angiotensin II (AngII) promotes hypertension and atherosclerosis by activating growth-promoting and pro-inflammatory gene expression in vascular smooth muscle cells (VSMCs). Enhancers and super-enhancers (SEs) play critical roles in driving disease-associated gene expression. However, enhancers/SEs mediating VSMC dysfunction remain uncharacterized. Here, we show that AngII alters vascular enhancer and SE repertoires in cultured VSMCs in vitro, ex vivo, and in AngII-infused mice aortas in vivo. AngII-induced enhancers/SEs are enriched in binding sites for signal-dependent transcription factors and dependent on key signaling kinases. Moreover, CRISPR-Cas9-mediated deletion of candidate enhancers/SEs, targeting SEs with the bromodomain and extra-terminal domain inhibitor JQ1, or knockdown of overlapping long noncoding RNAs (lncRNAs) blocks AngII-induced genes associated with growth-factor signaling and atherosclerosis. Furthermore, JQ1 ameliorates AngII-induced hypertension, medial hypertrophy and inflammation in vivo in mice. These results demonstrate AngII-induced signals integrate enhancers/SEs and lncRNAs to increase expression of genes involved in VSMC dysfunction, and could uncover novel therapies.